Belén Alonso

Espectro mutacional de los genes sarcoméricos MYH7, MYBPC3, TNNT2, TNNI3 y TPM1 en pacientes con miocardiopatía hipertrófica ☆

Introduccion y objetivos Las mutaciones en los genes sarcomericos son la causa mas frecuente de miocardiopatia hipertrofica. Para cada gen, la frecuencia de mutaciones varia entre los estudios, y las manifestaciones clinicas son muy heterogeneas, lo que dificulta el empleo de la informacion genetica en la practica clinica. Nuestro objetivo es determinar la frecuencia de mutaciones en los genes sarcomericos MYH7, MYBPC3, TNNT2, TNNI3 y TPM1 en una serie de pacientes con miocardiopatia hipertrofica. Metodos Se analizaron las regiones codificantes de estos cinco genes mediante secuenciacion en 120 pacientes (el 29% con antecedentes familiares), comparando el fenotipo segun el gen mutado. Resultados Se hallaron mutaciones en 32 pacientes; 10 y 20 tenian mutaciones en MYH7 (8%) y MYBPC3 (16%). Se hallaron mutaciones de TNNT2 y TPM1 en 2 y 1 pacientes, y ninguna de TNNI3. Dos pacientes tenian dos mutaciones (dobles mutantes). El 61% de las mutaciones no habian sido descritas previamente. No hallamos diferencias en la media de edad al diagnostico o el tamano de la hipertrofia entre los portadores de mutaciones en MYH7 y los de MYBPC3. Conclusiones El 26% de los pacientes tenian mutaciones en alguno de los cinco genes estudiados. Mas de la mitad de las mutaciones no habian sido descritas. El gen MYBPC3 fue el mas mutado, seguido de MYH7. No se hallaron diferencias fenotipicas entre los pacientes segun el gen mutado, lo que dificultaria el empleo de la informacion genetica para estratificar el riesgo en estos pacientes.

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia.

BackgroundHereditary Spastic Paraplegias (HSP) are characterized by progressive spasticity and weakness of the lower limbs. At least 45 loci have been identified in families with autosomal dominant (AD), autosomal recessive (AR), or X-linked hereditary patterns. Mutations in the SPAST (SPG4) and ATL1 (SPG3A) genes would account for about 50% of the ADHSP cases.MethodsWe defined the SPAST and ATL1 mutational spectrum in a total of 370 unrelated HSP index cases from Spain (83% with a pure phenotype).ResultsWe found 50 SPAST mutations (including two large deletions) in 54 patients and 7 ATL1 mutations in 11 patients. A total of 33 of the SPAST and 3 of the ATL1 were new mutations. A total of 141 (31%) were familial cases, and we found a higher frequency of mutation carriers among these compared to apparently sporadic cases (38% vs. 5%). Five of the SPAST mutations were predicted to affect the pre-mRNA splicing, and in 4 of them we demonstrated this effect at the cDNA level. In addition to large deletions, splicing, frameshifting, and missense mutations, we also found a nucleotide change in the stop codon that would result in a larger ORF.ConclusionsIn a large cohort of Spanish patients with spastic paraplegia, SPAST and ATL1 mutations were found in 15% of the cases. These mutations were more frequent in familial cases (compared to sporadic), and were associated with heterogeneous clinical manifestations.

Mutations in Sarcomeric Genes MYH7, MYBPC3, TNNT2, TNNI3, and TPM1 in Patients With Hypertrophic Cardiomyopathy

INTRODUCTION AND OBJECTIVES Mutation of a sarcomeric gene is the most frequent cause of hypertrophic cardiomyopathy. For each such gene, however, previous studies have reported a range of different mutation frequencies, and clinical manifestations have been highly heterogeneous, both of which limit the use of genetic information in clinical practice. Our aim was to determine the frequency of mutations in the sarcomeric genes MYH7, MYBPC3, TNNT2, TNNI3, and TPM1 in a cohort of Spanish patients with hypertrophic cardiomyopathy. METHODS We used sequencing to analyze the coding regions of these five genes in 120 patients (29% with a family history) and investigated how the patient phenotype varied with the gene mutated. RESULTS In total, 32 patients were found to have mutations: 10 in MYH7 (8%), 20 in MYBPC3 (16%), 2 in TNNT2, 1 in TPM1 and none in TNNI3. Overall, 61% of mutations had not been described before. Two patients had two mutations (i.e., double mutants). There was no difference in the mean age at diagnosis or the extent of the hypertrophy between those with MYH7 mutations and those with MYBPC3 mutations. CONCLUSIONS Some 26% of patients had a mutation in one of the five sarcomeric genes investigated. More than half of the mutations had not been described before. The MYBPC3 gene was the most frequently mutated, followed by MYH7. No phenotypic differences were observed between carriers of the various mutations, which makes it difficult to use genetic information to stratify risk in these patients.

Mitochondrial DNA and TFAM gene variation in early-onset myocardial infarction: Evidence for an association to haplogroup H

The main objective of this research was to define the association between common mitochondrial DNA (mtDNA) polymorphisms and mitochondrial transcription A gene (TFAM) variants and myocardial infarction (MI) in patients with atherosclerotic diseased vessels. Ten mitochondrial polymorphisms that defined the nine common European haplogroups were genotyped in 500 male patients with early onset MI (<55 years) and at least one atherosclerotic coronary vessel (angiographically confirmed), and 500 healthy controls. In addition, we searched for DNA variants in the coding region of the TFAM gene and compared patients and controls for the allele and genotype frequencies. Early onset MI was strongly associated with male gender and tobacco smoking in our population. MtDNA haplogroup H (defined by allele 7028 °C) was significantly more frequent in a first group of patients (n = 250) compared to controls (n = 300), and the association was confirmed in a second group of only smokers (250 patients and 200 controls). For total patients and controls, we obtained a p = 0.002 (OR = 1.50; 95% CI = 1.17-1.92) for H vs. the other haplogroups. We found four common TFAM polymorphisms, with allele/genotype frequencies that did not differ between patients and controls. In conclusion, mitochondrial haplogroup H was associated with early onset MI in male smokers. Our work supported a role for the mtDNA variation in the risk for atherosclerosis and ischemic associated events, likely due to differences in mitochondrial function and reactive oxygen production between the different haplogroups.

Late-onset Alzheimer's disease is associated with mitochondrial DNA 7028C/haplogroup H and D310 poly-C tract heteroplasmy

There is growing evidence that mitochondria play an important role in the pathogenesis of late-onset Alzheimers disease (LOAD) [1]. Certain haplogroups defined by combinations of common mitochondrial DNA (mtDNA) single-nucleotide polymorphisms (SNPs) have been associated with longevity and age-related diseases. Recent studies reported a significant association between haplogroup H and LOAD [2–4] (supplementary Table 1). Differences between the mtDNA SNPs/haplogroups in the efficiency of oxygen consumption and ATP and ROS production could partly explain these associations [5]. Although the relative risk attributed to this haplogroup was low (<1.5) in all the studies, this could have important implications considering the high incidence of LOAD at the population level. It is therefore very important to determine the influence of mtDNA polymorphisms in LOAD by studying large cohorts from different populations. We report the results from Asturias (northern Spain) based on a total of 500 LOAD patients and 500 controls (Supplementary file). The mtDNA 7028C was significantly more frequent among the patients (p=0.001; OR=1.52, 95% CI=1.18– 1.95; Table 1). Allele frequencies for the other six SNPs that defined the mitochondrial haplogroups did not differ between patients and controls (data not shown). Haplogroup H (defined by 7028C) was significantly more frequent among the patients (Supplementary file). Multiple logistic regression with apolipoprotein E gene (APOE)-ε4 and mtDNA 7028C status, age, and gender as covariates showed that APOE-ε4 and 7028C were associated with LOAD. We found a synergistic effect between the two risk variants: the OR for APOE-ε4 + 7028C (OR=5.30, 95% CI=3.72–7.54) was higher than the OR for each separately. The non-APOE-ε4 + 7028T had a protective effect (OR= 0.43; 95%CI=0.33–0.75). Patients and controls were classified as D310 7C or D310 >7C (poly-C tract at position 310 of the mtDNA, Supplementary file). We found a significantly higher frequency of the >7C alleles among the patients (Table 1). Electronic supplementary material The online version of this article (doi:10.1007/s10048-011-0295-4) contains supplementary material, which is available to authorized users. E. Coto (*) : J. Gómez :B. Alonso :A. I. Corao :M. Díaz : V. Álvarez Genética Molecular-Laboratorio de Medicina, Hospital Universitario Central Asturias, 33006 Oviedo, Spain e-mail: eliecer.coto@sespa.princast.es

Screening of the Filamin C Gene in a Large Cohort of Hypertrophic Cardiomyopathy PatientsCLINICAL PERSPECTIVE

Background— Recent exome sequencing studies identified filamin C (FLNC) as a candidate gene for hypertrophic cardiomyopathy (HCM). Our aim was to determine the rate of FLNC candidate variants in a large cohort of HCM patients who were also sequenced for the main sarcomere genes. Methods and Results— A total of 448 HCM patients were next generation–sequenced (semiconductor chip technology) for the MYH7, MYBPC3, TNNT2, TNNI3, ACTC1, TNNC1, MYL2, MYL3, TPM1, and FLNC genes. We also sequenced 450 healthy controls from the same population. Based on the reported population frequencies, bioinformatic criteria, and familial segregation, we identified 20 FLNC candidate variants (13 new; 1 nonsense; and 19 missense) in 22 patients. Compared with the patients, only 1 of the control’s missense variants was nonreported (P=0.007; Fisher exact probability test). Based on the familial segregation and the reported functional studies, 6 of the candidate variants (in 7 patients) were finally classified as likely pathogenic, 10 as variants of uncertain significance, and 4 as likely benign. Conclusions— We provide a compelling evidence of the involvement of FLNC in the development of HCM. Most of the FLNC variants were associated with mild forms of HCM and a reduced penetrance, with few affected in the families to confirm the segregation. Our work, together with others who found FLNC variants among patients with dilated and restrictive cardiomyopathies, pointed to this gene as an important cause of structural cardiomyopathies.

Lack of association between protocadherin 11-X/Y (PCDH11X and PCDH11Y) polymorphisms and late onset Alzheimer's disease.

A recent genome-wide association study (GWA) reported a significant association between single nucleotide polymorphisms (SNPs) at the PCDH11X gene and late-onset Alzheimers disease (LOAD). Our research was designated to replicate this association, including non previously analyzed PCDH11X and PCDH11Y SNPs. We genotyped four PCDH11X and one PCDH11Y SNPs in a total of 420 LOAD patients and 350 healthy controls from Spain. Allele and genotype frequencies did not differ between patients and controls for the five SNPs, even after correcting by gender, age, and APOE-ε4 status. Our data were in agreement with recent reports that failed to confirm the association between PCDH11X polymorphisms and LOAD, and extended the lack of association to common PCDH11Y variants.

A labor and cost effective next generation sequencing of PKHD1 in autosomal recessive polycystic kidney disease patients

The Sanger sequencing of patients with recessive polycystic kidney disease is challenging due to the length and heterogeneous mutational spectrum of the PKHD1 gene. Next generation sequencing (NGS) might thus be of special interest to search for PKHD1 mutations. The study involved a total of 22 patients with autosomal recessive polycystic kidney disease (ARPKD) and 8 parents of non-available ARPKD patients. Five pools of 6 samples each were sequenced with the Personal Genome Machine (PGM, Ion Torrent). For each DNA pool, a total of 109 fragments that covered the entire PKHD1 coding sequence were amplified in only two tubes followed by library preparation and NGS with the PGM. To validate the technique, each pool contained the DNA of at least one patient with known mutation. The putative mutations identified in each pool were confirmed and assigned to specific individuals through Sanger sequencing. All but one of the 109 amplicons were successfully read, and we identified the two PKHD1 mutations in 11 of the ARPKD cases, one mutation in 9 patients, and no mutation in only 2 patients. Six of the 8 parents from non-available patients were mutation carriers. The reported procedure would facilitate the large scale analysis of PKHD1 with a significant reduction in cost and labor.

Amyloid Precursor Protein Gene (APP) Variation in Late-Onset Alzheimer’s Disease

Mutations in the beta-amyloid precursor protein gene (APP) have been found in familial early-onset Alzheímer’s disease (AD). DNA variants at several genes have been linked to the risk of developing the most common late-onset form of AD (LOAD). A few studies analyzed the contribution of APP variants to LOAD, with negative or conflicting results. We determined the variation in the 18 APP exons and flanking intronic sequences in a total of 350 LOAD patients from Spain. A total of 13 nucleotide changes were found and 6 were new and not found among 340 healthy controls, including the only missense change (D243N). The in silico analysis suggested that none of them would have an effect on pre-mRNA splicing or protein folding (D243N). Patients and controls were also genotyped for three APP promoter polymorphisms, and none of them was significantly associated with LOAD. We concluded that APP variants would not contribute to the risk of developing LOAD in our population.

ABCB1 (MDR-1) pharmacogenetics of tacrolimus in renal transplanted patients: a Next Generation Sequencing approach

Abstract Background: A CYP3A5 gene polymorphism is the main determinant of Tacrolimus (Tac) dose requirements among renal transplanted patients. In spite of the utility of CYP3A5 genotyping to predict the Tac-dose, many patients exhibit an out of range blood Tac level and it is thus likely that other genes/polymorphisms contribute to define Tac bioavailability. To address this issue we searched for coding sequence variants in the ABCB1/MDR1 gene in renal transplanted patients treated with Tac and who had out of the range blood levels. Methods: We studied 100 renal transplanted patients treated with Tac, 60 of whom had Tac blood levels below (n=39) and above (n=21) the target range (10–15 ng/mL) at 1 week post-transplant. The DNA was subjected to multiplex amplification followed by massive parallel semiconductor sequencing in the Ion Torrent personal genome machine. Results: We found four missense changes, all reported and present in cases above and below the blood Tac target. In addition, we did not find differences in the allele and genotype frequencies for the common rs1045642 polymorphism (p.I1145I) between the groups. Conclusions: Our results suggested that the ABCB1 variants had no effect on the risk of showing out of range Tac blood levels among renal transplanted patients.