Compound heterozygosity for PTPN11 variants in a subject with Noonan syndrome provides insights into the mechanism of SHP2-related disorders.

The RASopathies are a family of clinically related disorders caused by mutations affecting genes participating in the RAS-MAPK signaling cascade. Among them, Noonan syndrome (NS) and Noonan syndrome with multiple lentigines (NSML) are allelic conditions principally associated with dominant mutations in PTPN11, which encodes the nonreceptor SH2 domain-containing protein tyrosine phosphatase SHP2. Individual PTPN11 mutations are specific to each syndrome and have opposite consequences on catalysis, but all favor SHP2's interaction with signaling partners. Here, we report on a subject with NS harboring biallelic variants in PTPN11. While the former (p.Leu261Phe) had previously been reported in NS, the latter (p.Thr357Met) is a novel change impairing catalysis. Members of the family carrying p.Thr357Met, however, did not show any obvious feature fitting NSML or within the RASopathy phenotypic spectrum. A major impact of this change on transcript processing and protein stability was excluded. These findings further support the view that NSML cannot be ascribed merely to impaired SHP2's catalytic activity and suggest that PTPN11 mutations causing this condition act through an alternative dominant mechanism.

The APOB polymorphism rs1801701 A/G (p.R3638Q) is an independent risk factor for early-onset coronary artery disease: Data from a Spanish cohort.

BACKGROUND AND AIMS: Apoliprotein B (ApoB) has been associated with hypercholesterolemia and ischemic coronary disease. This study was aimed to determine the effect of two APOB gene variants in the risk of developing early-onset coronary artery disease (EO-CAD) in a Spanish population. The association of these polymorphisms with hypercholesterolemia was also analysed. METHODS AND RESULTS: The study involved a total of 889 healthy population controls (397 male) and 790 EO-CAD cases (636 male; EO-CAD was defined as male <60 years and women <65 years). All the patients had at least one vessel with angiography documented atherosclerotic lesion. Patients and controls were genotyped for the APOB variants rs1801701 A/G (p.R3638Q) and rs1367117 C/T (p.T98I). Allele and genotype frequencies were compared between the groups (patients vs. controls, hyper-vs. normo-cholesterolemia) by logistic regression. The rs1801701 was significantly associated with EO-CAD in male (OR = 1.44, 95%CI = 1.05-1.99) and female (OR = 2.22, 95%CI = 1.58-3.14). This SNP was significantly associated with hypercholesterolemia in female, with a trend in male. The association with EO-CAD was independent of hypercholesterolemia (multiple logistic regression). CONCLUSION: A common APOB polymorphism (rs1801701) was an independent risk factor for EO-CAD in our population. The risk-effect was more significant in female than in male.

IL17RA in early-onset coronary artery disease: Total leukocyte transcript analysis and promoter polymorphism (rs4819554) association.

BACKGROUND AND AIMS: The interleukin-17 (IL-17) pathway would play an important role in the pathogenesis of atherosclerosis and coronary-artery disease (CAD). The IL-17 inflammatory mediators are expressed by Th17 cells, a group of CD4 + leukocytes that infiltrate the vascular milieu and are pivotal in the origin, progression, stability and rupture of the atherosclerotic lesion. Cigarette smoke compounds stimulated the expression of IL-17 and IL-17-receptors. In atherogenic mice models the deficiency of IL-17RA resulted in a reduction of the atherosclerotic lesion size and leukocyte infiltrate. We hypothesised that common the IL-17RA transcript might be differential expressed in the leukocytes from CAD patients and healthy individuals. METHODS: The relative amount of the IL-17RA to ACTB transcript was determined in total leukocytes of 55 patients and 50 controls, all smokers. We genotyped the IL-17RA rs48195554 promoter polymorphisms in 390 healthy controls and 450 early-onset CAD patients. RESULTS: Patients showed significantly higher mean IL-17RA normalised transcript value than controls (p < 0.001). For the IL-17RA rs48195554 promoter polymorphisms, IL-17RA G-carriers showed higher transcript values. However, allele and genotype frequencies did not differ between patients and controls and we thus excluded a significant association with CAD. CONCLUSIONS: The higher levels of the IL-17RA transcript among CAD-patients was in agreement with a role for the IL-17 pathway in the pathogenesis of coronary atherosclerosis.

Role of syncope in predicting adverse outcomes in patients with suspected Brugada syndrome undergoing standardized flecainide testing.

Aims: Sensitivity to flecainide testing results in suboptimal findings in patients with Brugada syndrome (BrS), leading to safety concerns. Because cardiac syncope effectively predicts outcomes in BrS, we aimed to explore its predictive value in a large cohort of negative and positive responders (NR and PR) to standard flecainide testing. Methods and results: We analysed the data of 251 consecutive patients, 177 NR vs. 74 PR, to flecainide testing, performed according to standard recommendations. Cardiac syncope was defined as syncope presenting without prodromal symptoms and in the absence of any specific situation. Comparing PR with NR, there were no differences regarding age (39 ± 15 vs. 44 ± 13 years; P = 0.052), male gender (70.1% vs. 66.2%; P = 0.553), and family history of sudden cardiac death in relatives younger than 45 years (27% vs. 27%; P = 1). Cardiac syncope was more frequent in PR (12.2% vs. 4%; P = 0.022), and previous sudden cardiac arrest (SCA) was documented only in PR (5.4% vs. 0%; P = 0.007). During the follow-up period (6.2 ± 3.3 years), one NR, who had previously experienced cardiac syncope, developed SCA 3 months after flecainide testing. Following resuscitation, a type I electrocardiogram was spontaneously recorded. The follow-up event rate was higher in patients with cardiac syncope, both in PR and in NR (P < 0.001 both). In a multivariate analysis, cardiac syncope was the unique variable that predicted adverse outcomes (hazard ratio 14.9, 95% confidence interval 1.84-121.25; P = 0.011). Conclusions: In patients with false-negative responses to the provocative testing with flecainide, cardiac syncope predicts SCA, allowing a more extensive and individualized evaluation.

A Semiconductor Chip-Based Next Generation Sequencing Procedure for the Main Pulmonary Hypertension Genes.

The aim of this study was to characterize the mutational spectrum of pulmonary hypertension (PH) patients through a next generation sequencing platform. In a total of 22 patients, the BMPR2, SMAD9, CAV1, KCNK3, and EIF2AK4 genes were sequenced with semiconductor chips and the ion torrent personal genome machine. We found six putative mutations in SMAD (p.R263Q), BMPR2 (p.S301P, p.T493I), CAV1 (p.V155I), and EIF2AK4 (p.L489P, p.P1115L) in five patients. One patient was compound heterozygous for BMPR2 + SMAD mutations, and one patient was homozygous for EIF2AK4 p.P1115L. The reported procedure would facilitate the rapid mutational screening of large cohorts of PH patients.

Mutation analysis of the main hypertrophic cardiomyopathy genes using multiplex amplification and semiconductor next-generation sequencing.

BACKGROUND: Mutations in at least 30 genes have been linked to hypertrophic cardiomyopathy (HCM). Due to the large size of the main HCM genes, Sanger sequencing is labor intensive and expensive. The purpose was to develop a next-generation sequencing (NGS) procedure for the main HCM genes. METHODS AND RESULTS: Multiplex amplification of the coding exons of MYH7,MYBPC3,TNNT2,TNNI3,ACTC1,TNNC1,MYL2,MYL3, and TPM1 was designated, followed by NGS with the Ion Torrent PGM (Life Technologies). A total of 8 pools containing DNA from HCM patients were sequenced in a 2-step approach. First, a total of 60 patients (validation cohort) underwent both PGM and Sanger sequencing for the 9 genes. No false-negative variants were found on NGS (100% sensitivity), and a specificity of 97% and 80% was achieved for single-nucleotide and insertion/deletion variants, respectively. Second, the PGM was used to search for mutations in a total of 76 cases not previously studied (discovery cohort). A total of 19 putative mutations were identified in the discovery pools, which were confirmed and assigned to specific patients on Sanger sequencing. CONCLUSIONS: An NGS procedure has been developed for the main sarcomeric genes that would facilitate the screening of large cohorts of patients. In addition, this procedure would facilitate the uncovering of rare gene variants on a population scale.

New polymorphisms in human MEF2C gene as potential modifier of hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is caused by mutations in genes encoding sarcomeric proteins. Its variable phenotype suggests the existence of modifier genes. Myocyte enhancer factor (MEF) 2C could be important in this process given its role as transcriptional regulator of several cardiac genes. Any variant affecting MEF2C expression and/or function may impact on hypertrophic cardiomyopathy clinical manifestations. In this candidate gene approach, we screened 209 Caucasian hypertrophic cardiomyopathy patients and 313 healthy controls for genetic variants in MEF2C gene by single-strand conformation polymorphism analysis and direct sequencing. Functional analyses were performed with transient transfections of luciferase reporter constructions. Three new variants in non-coding exon 1 were found both in patients and controls with similar frequencies. One-way ANOVA analyses showed a greater left ventricular outflow tract obstruction (p = 0.011) in patients with 10C+10C genotype of the c.-450C(8_10) variant. Moreover, one patient was heterozygous for two rare variants simultaneously. This patient presented thicker left ventricular wall than her relatives carrying the same sarcomeric mutation. In vitro assays additionally showed a slightly increased transcriptional activity for both rare MEF2C alleles. In conclusion, our data suggest that 15 bp-deletion and C-insertion in the 5'UTR region of MEF2C could affect hypertrophic cardiomyopathy, potentially by affecting expression of MEF2C and therefore, the expression of their target cardiac proteins that are implicated in the hypertrophic process.

Association of the Genetic Variation in the Long Non-Coding RNA FENDRR with the Risk of Developing Hypertrophic Cardiomyopathy.

Background: In around 40−60% of Hypertrophic Cardiomyopathy (HCM) cases pathogenic variants are not identified. Our aim was to evaluate the possible association of lncRNAs with the risk of developing HCM. Methods: We sequenced 10 lncRNAs coding genes that have been associated with cardiovascular disease in a discovery cohort (238 HCM patients and 212 controls) by NGS, and genotyped rs74035787 G>A and rs1424019 A>G polymorphism in a validation cohort (962 HCM patients and 923 controls). Finally, we sequenced the FENDRR promoter by Sanger sequencing. Results: We observed by NGS that FENDRR rs39527, rs39529 and rs40384 polymorphisms were significantly associated with HCM in our cohort (p = 0.0284; OR: 0.24, 95%CI: 0.07−0.86). NGS results were confirmed by genotyping rs74035787 polymorphism (p = 0.001; OR:0.38, 95%CI: 0.21−0.66). Moreover, it is also associated when stratification by sex (p = 0.003; OR:0.20, 95%CI: 0.06−0.53), and age (≥50 years old p = 0.001, OR:0.33, 95%CI: 0.16−0.63) Moreover, the risk of HCM in the carriers of the GG genotype of the rs1424019 polymorphism was significantly higher than that of the AA/AG genotypes carriers in the elderly subjects (p = 0.045, OR:1.24, 95%CI: 1.01−1.53). On the other hand, we observed significant differences in the rs74035787 A/rs1424019 G haplotype frequency (p = 0.0035; OR: 0.20, 95%CI: 0.07−0.59). Conclusions: Our study suggested a significant association between FENDRR gene variants and HCM.

Functional polymorphisms in genes of the Angiotensin and Serotonin systems and risk of hypertrophic cardiomyopathy: AT1R as a potential modifier.

BACKGROUND: Angiotensin and serotonin have been identified as inducers of cardiac hypertrophy. DNA polymorphisms at the genes encoding components of the angiotensin and serotonin systems have been associated with the risk of developing cardiovascular diseases, including left ventricular hypertrophy (LVH). METHODS: We genotyped five polymorphisms of the AGT, ACE, AT1R, 5-HT2A, and 5-HTT genes in 245 patients with Hypertrophic Cardiomyopathy (HCM; 205 without an identified sarcomeric gene mutation), in 145 patients with LVH secondary to hypertension, and 300 healthy controls. RESULTS: We found a significantly higher frequency of AT1R 1166 C carriers (CC+AC) among the HCM patients without sarcomeric mutations compared to controls (p = 0.015; OR = 1.56; 95%CI = 1.09-2.23). The AT1R 1166 C was also more frequent among patients who had at least one affected relative, compared to sporadic cases. This allele was also associated with higher left ventricular wall thickness in both, HCM patients with and without sarcomeric mutations. CONCLUSIONS: The 1166 C AT1R allele could be a risk factor for cardiac hypertrophy in patients without sarcomeric mutations. Other variants at the AGT, ACE, 5-HT2A and 5-HTT did not contribute to the risk of cardiac hypertrophy.

Congenital long QT syndrome: Should citric fruits be avoided? A case report of a cLQTS type 1 with unusual QTc prolongation with citric juices.

Congenital long QT syndromes (cLQTS) are relatively rare diseases in which QT interval is prolonged due to several mutations on ion channels involved in cardiac cell repolarization. This condition confers higher risk of malignant arrhythmias and sudden cardiac death, and it is widely accepted that substances that prolong QT interval should be avoided by these patients. Most of these substances are antibiotics and non-antibiotics drugs, but almost nothing is known about frequently consumed fruits and juices. We report the case of a patient with a previously asymptomatic cLQTS type 1 (cLQTS1) with unusual QT prolongation of 167 milliseconds (ms) related to the consumption of large amounts of citric juices (oranges and lemons). A literature review was done for better understanding of its influence on QT interval duration and to know the concentration of flavonoids on citric fruits. .

Gene variants in the NF-KB pathway (NFKB1, NFKBIA, NFKBIZ) and risk for early-onset coronary artery disease.

The nuclear-factor kappa-beta (NF-KB) is a driver of inflammation, and plays an important role in the pathogenesis of atherosclerosis and coronary artery disease (CAD). Early-onset CAD is defined as a coronary ischaemic episode at an age ≤55 years, and in our population was strongly associated with male sex and smoking. Our aim was to determine whether common variants in three NF-KB genes were associated with early-onset CAD. We studied 609 patients with early-onset CAD and 423 healthy controls, all male. Allele and genotype frequencies for the NFKB1 rs28362491 (-94 delATTG) and NFKBIA rs8904 were not significantly different between the two groups. For the NFKBIZ rs3217713, the deletion allele was significantly more frequent in the patients than in controls (0.27 vs. 0.22; p = 0.004). Deletion-carriers were more frequent in the patients (p < 0.001), with an OR = 1.48 (95%CI = 1.15-1.90). We performed a multiple logistic regression (linear generalized model) with smoking, hypercholesterolemia, type 2 diabetes, hypertension, and the rs3217713 deletion carriers remained significantly associated with early-onset CAD (p = 0.01). In our population, the NFKBIZ variant was an independent risk factor for developing early-onset CAD.

Spectral Analysis of the QT Interval Increases the Prediction Accuracy of Clinical Variables in Brugada Syndrome.

(1) Background: The clinical management of Brugada Syndrome (BrS) remains suboptimal. (2) Objective: To explore the role of standard electrocardiogram (ECG) spectral analysis in diagnosis and risk stratification. (3) Methods: We analyzed 337 patients-43 with a spontaneous type I ECG pattern (Spont-BrS), 112 drug induced (Induct-BrS), and 182 with a negative response to the drug challenge (negative responders (NR)). ECGs were processed using the wavelet transform (high frequency: 85 to 130 Hz). (4) Results: The power of the high-frequency content in the ST segment (Total ST Power; nV²Hz-¹10³) was higher in BrS compared with NR patients (Spont-BrS: 28.126 (7.274-48.978) vs. Induc-BrS: 26.635 (15.846-37.424) vs. NR: 11.13 (8.917-13.343); p = 0.002). No differences were observed between ECG patterns in BrS patients. However, the Total ST Power of the type II or III ECG in NR patients was lower than in the same ECG patterns recorded from BrS patients (BrS: 31.07 (16.856-45.283); vs. NR: 10.8 (7.248-14.352) nV²Hz-¹10³; p = 0.007). The Total ST Power, age, and family history of BrS were independent predictors of positive responses to drug testing. Comparing models with versus those without Total ST Power, the area under the received operator curve (ROC) curve increased (with 0.607 vs. without 0.528, p = 0.001). Only syncope was associated with an increased risk (follow-up 55.8 ± 39.35 months). However, the area under the ROC curve increased significantly when the Total ST Power was included as a covariate (with 0.784 vs. without 0.715, p = 0.04). (5) Conclusions: The analysis of the high-frequency content of ECG signals increases the predictive capability of clinical variables in BrS patients.

Mitochondrial transcription factors TFA, TFB1 and TFB2: a search for DNA variants/haplotypes and the risk of cardiac hypertrophy.

Mitochondrial transcription factors mtTFA, mtTFB1 and mtTFB2 are required for the replication of mitochondrial DNA (mtDNA), regulating the number of mtDNA copies. Mice with a mtTFA deletion showed a reduced number of mtDNA copies, a reduction in respiratory chain activity, and a characteristic dilated cardiomyopathy. DNA variants in these genes could be involved in the risk for cardiac hypertrophy (HCM). We determined the variation in the TFAM, TFB1M, and TFB2M genes (using SSCA, DHPLC, and direct sequencing) in a total of 200 HCM-patients from Spain and Germany, and in 250 healthy controls. We found several common polymorphisms that defined haplotype blocks in these genes, with frequencies that did not differ between patients and controls. We also found four novel variants in patients which were absent in the controls: -91 C > A (5'-UTR) and Ala105 > Thr in TFAM, and Thr211 > Ala and Arg256 > Lys in TFB1M. The three missense changes were in highly conserved amino acids, and could be involved in HCM-risk. In conclusion, common variants in the mitochondrial transcription factors were not associated with the risk for HCM. However, rare DNA variants (putative mutations) could be involved in the pathogenesis of HCM in a reduced number of cases.

Gene variants in the NF-KB pathway (NFKB1, NFKBIA, NFKBIZ) and their association with type 2 diabetes and impaired renal function.

The NF-kappaB pathway might play a role in the pathogenesis of renal disease and type 2 diabetes (T2DM). Our aim was to determine whether common polymorphisms in NF-kappaB genes were associated with impaired renal function and T2DM in a cohort of healthy elderly individuals. We studied 487 individuals, all Caucasian and aged 65-85 years. A total of 104 (21%) had impaired renal function (estimated glomerular filtration rate, eGFR < 60) and 146 (30%) were classified as diabetics. The genotypes of 4 common variants were determined through PCR-RFLP or fluorescent capillary electrophoresis. The NFKB1 variants were significantly associated with T2DM: rs7667496 p = 0.01, OR = 1.68; and rs28362491 p = 0.02, OR = 1.67. They remained significantly associated in a multiple logistic regression with age, gender, hypertension, body mass index, and cholesterol. There was a trend toward the association of these variants with eGFR < 60. The two NFKB1 variants were in linkage disequilibrium (D' = -0.86), and homozygous for the two non-risk alleles (rs7667496 CC + rs28362491 II), were significantly more common in the non-diabetics (p = 0.02). In our cohort the NFKB1 variation was an independent risk factor for developing T2DM. Additional studies to confirm this association are of special interest, as well as studies to give a functional explanation to the genetic association.

Genetic Variation in the H19-IGF2 Cluster Might Confer Risk of Developing Impaired Renal Function.

The H19-IGF2 imprinted gene region could be implicated in the risk of developing impaired renal function (IRF). Our aim was to determine the association of several common H19-IGF2 variants and IRF in a cohort of elderly healthy individuals. The study involved 675 individuals >65 years of age, 184 with type 2 diabetes mellitus (T2DM), and 105 with IRF (estimated glomerular filtration rate [eGFR] <60). They were genotyped for two common H19 single nucleotide polymorphisms (SNPs) (rs2839698 and rs10732516), one H19-IGF2 intergenic indel (rs201858505), and one indel in the 3'UTR of the IGF2. For the H19 SNPs, we also determined the allele present in the methylated chromosome through genotyping the DNA digested with a methylation-sensitive endonuclease. None of the four H19-IGF2 variants was associated with IRF in our cohort. We found a significantly higher frequency of the 3'UTR IGF2 deletion (D) in the eGFR <60 group (p = 0.01; odds ratio = 1.16, 95% confidence interval = 1.10-2.51). This association was independent of age and T2DM, two strong predictors of IRF. In conclusion, a common indel variant in the 3'UTR of the IGF2 gene was associated with the risk of IRF. This association could be explained by the role of IGF2 in podocyte survival, through regulation of IGF2 expression by differential binding of miRNAs to the indel sequences. Functional studies should be necessary to clarify this issue.

Genetic variation at the long noncoding RNA H19 gene is associated with the risk of hypertrophic cardiomyopathy.

AIM: The long noncoding RNA H19 and its host micro RNA miR-675 have been found deregulated in cardiac hypertrophy and heart failure tissues. Our aim was to investigate whether the H19 gene variants were associated with the risk of hypertrophic cardiomyopathy (HCM). PATIENTS & METHODS: We genotyped two H19 tag single nucleotide polymorphisms in 405 HCM patients and 550 controls, and sequenced this gene in 100 patients. RESULTS: The rs2107425 C was significantly increased in sarcomere no-mutation patients (n = 225; p = 0.01): CC versus CT + TT, p = 0.017; odd ratios: 1.51. Sequencing of the H19 coding transcript identified two patients heterozygous carriers for a rare variant, rs945977096 G/A, that was absent among the controls. CONCLUSION: Our study suggested a significant association between H19 variants and the risk of developing HCM.

Mitochondrial haplogroup T is negatively associated with the status of elite endurance athlete.

Mitochondrial function is absolutely necessary to supply the energy required for muscles, and germ line mutations in mitochondrial genes have been related with impaired cardiac function and exercise intolerance. In addition, alleles at several polymorphic sites in mtDNA define nine common haplogroups, and some of these haplogroups have been implicated in the risk of developing several diseases. In this study, we analysed the association between mtHaplogroups and the capacity to reach the status of elite endurance athlete. DNA was obtained from blood leukocytes of 95 Spanish elite endurance athletes and 250 healthy male population controls. We analysed eight mitochondrial polymorphisms and the frequencies were statistically compared between elite athletes and controls. Haplogroup T, specifically defined by 13368A, was significantly less frequent among elite endurance athletes (p =0.012, Fisher's exact test). Our study suggests that allele 13368A and mitochondrial haplogroup T might be a marker negatively associated with the status of elite endurance athlete. This mitochondrial variant could be related with a lower capacity to respond to endurance training, through unknown mechanisms involving a less efficient mitochondrial workload.

Differential methylation of lncRNA KCNQ1OT1 promoter polymorphism was associated with symptomatic cardiac long QT.

AIM: To investigate whether the differential methylation of KCNQ1OT1 was associated with the risk of symptomatic long QTc. PATIENTS & METHODS: We investigated the methylation status of KCNQ1OT1 in a cohort of patients (n = 131) with a symptomatic prolonged QTc. All the patients were genotyped for a common promoter polymorphism (rs11023840). They were also genotyped for DNA digested with the methylation-sensitive HpaII restriction enzyme. RESULTS: We found a significant higher frequency of AA genotype (p = 0.02) in the patients compared with healthy controls (n = 240). In the HpaII-digested samples there was a higher frequency of the A-allele among the patients compared with the controls (p = 0.02). CONCLUSION: Our findings supported a role for the differential methylation/imprinting of KCNQ1OT1 in the risk for symptomatic prolonged QTc.

Screening of the Filamin C Gene in a Large Cohort of Hypertrophic Cardiomyopathy Patients.

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.