DNA-pools targeted-sequencing as a robust cost-effective method to detect rare variants: Application to dilated cardiomyopathy genetic diagnosis.

Dilated cardiomyopathy (DCM) is a heart disease characterized by left ventricular dilatation and systolic dysfunction. In 30% of cases, pathogenic variants, essentially private to each patient, are identified in at least one of almost 50 reported genes. Thus, while costly, exons capture-based Next Generation Sequencing (NGS) of a targeted gene panel appears as the best strategy to genetically diagnose DCM. Here, we report a NGS strategy applied to pools of 8 DNAs from DCM patients and validate its robustness for rare variants detection at 4-fold reduced cost. Our pipeline uses Freebayes to detect variants with the expected 1/16 allele frequency. From the whole set of detected rare variants in 96 pools we set the variants quality parameters optimizing true positives calling. When compared to simplex DNA sequencing in a shared subset of 50 DNAs, 96% of SNVs/InsDel were accurately identified in pools. Extended to the 384 DNAs included in the study, we detected 100 variants (ACMG class 4 and 5), mostly in well-known morbid gene causing DCM such as TTN, MYH7, FLNC, and TNNT2. To conclude, we report an original pool-sequencing NGS method accurately detecting rare variants. This innovative approach is cost-effective for genetic diagnostic in rare diseases.

Emery-Dreifuss muscular dystrophy Type 1 is associated with a high risk of malignant ventricular arrhythmias and end-stage heart failure.

BACKGROUND AND AIMS: Emery-Dreifuss muscular dystrophy (EDMD) is caused by variants in EMD (EDMD1) and LMNA (EDMD2). Cardiac conduction defects and atrial arrhythmia are common to both, but LMNA variants also cause end-stage heart failure (ESHF) and malignant ventricular arrhythmia (MVA). This study aimed to better characterize the cardiac complications of EMD variants. METHODS: Consecutively referred EMD variant-carriers were retrospectively recruited from 12 international cardiomyopathy units. MVA and ESHF incidences in male and female variant-carriers were determined. Male EMD variant-carriers with a cardiac phenotype at baseline (EMDCARDIAC) were compared with consecutively recruited male LMNA variant-carriers with a cardiac phenotype at baseline (LMNACARDIAC). RESULTS: Longitudinal follow-up data were available for 38 male and 21 female EMD variant-carriers [mean (SD) ages 33.4 (13.3) and 43.3 (16.8) years, respectively]. Nine (23.7%) males developed MVA and five (13.2%) developed ESHF during a median (inter-quartile range) follow-up of 65.0 (24.3-109.5) months. No female EMD variant-carrier had MVA or ESHF, but nine (42.8%) developed a cardiac phenotype at a median (inter-quartile range) age of 58.6 (53.2-60.4) years. Incidence rates for MVA were similar for EMDCARDIAC and LMNACARDIAC (4.8 and 6.6 per 100 person-years, respectively; log-rank P = .49). Incidence rates for ESHF were 2.4 and 5.9 per 100 person-years for EMDCARDIAC and LMNACARDIAC, respectively (log-rank P = .09). CONCLUSIONS: Male EMD variant-carriers have a risk of progressive heart failure and ventricular arrhythmias similar to that of male LMNA variant-carriers. Early implantable cardioverter defibrillator implantation and heart failure drug therapy should be considered in male EMD variant-carriers with cardiac disease.

Phenotype/Genotype Relationship in Left Ventricular Noncompaction: Ion Channel Gene Mutations Are Associated With Preserved Left Ventricular Systolic Function and Biventricular Noncompaction: Phenotype/Genotype of Noncompaction.

BACKGROUND: Few data exist concerning genotype-phenotype relationships in left ventricular noncompaction (LVNC). METHODS AND RESULTS: From a multicenter French Registry, we report the genetic and clinical spectrum of 95 patients with LVNC, and their genotype-phenotype relationship. Among the 95 LVNC, 45 had at least 1 mutation, including 14 cases of mutation in ion channel genes. In a complementary analysis including 16 additional patients with ion channel gene mutations, for a total of 30 patients with ion channel gene mutation, we found that those patients had higher median LV ejection fraction (60% vs 40%; P < .001) and more biventricular noncompaction (53.1% vs 18.5%; P < .001) than the 81 other patients with LVNC. Among them, both the 19 patients with an HCN4 mutation and the 11 patients with an RYR2 mutation presented with a higher LV ejection fraction and more frequent biventricular noncompaction than the 81 patients with LVNC but with no mutation in the ion channel gene, but only patients with HCN4 mutation presented with a lower heart rate. CONCLUSIONS: Ion channel gene mutations should be searched systematically in patients with LVNC associated with either bradycardia or biventricular noncompaction, particularly when LV systolic function is preserved. Identifying causative mutations is of utmost importance for genetic counselling of at-risk relatives of patients affected by LVNC.

FLNC pathogenic variants in patients with cardiomyopathies: Prevalence and genotype-phenotype correlations.

Pathogenic variants in FLNC encoding filamin C have been firstly reported to cause myopathies, and were recently linked to isolated cardiac phenotypes. Our aim was to estimate the prevalence of FLNC pathogenic variants in subtypes of cardiomyopathies and to study the relations between phenotype and genotype. DNAs from a cohort of 1150 unrelated index-patients with isolated cardiomyopathy (700 hypertrophic, 300 dilated, 50 restrictive cardiomyopathies, and 100 left ventricle non-compactions) have been sequenced on a custom panel of 51 cardiomyopathy disease-causing genes. An FLNC pathogenic variant was identified in 28 patients corresponding to a prevalence ranging from 1% to 8% depending on the cardiomyopathy subtype. Truncating variants were always identified in patients with dilated cardiomyopathy, while missense or in-frame indel variants were found in other phenotypes. A personal or family history of sudden cardiac death (SCD) was significantly higher in patients with truncating variants than in patients carrying missense variants (P = .01). This work reported the first observation of a left ventricular non-compaction associated with a unique probably causal variant in FLNC which highlights the role of FLNC in cardiomyopathies. A correlation between the nature of the variant and the cardiomyopathy subtype was observed as well as with SCD risk.

Prognosis of Adults With Isolated Left Ventricular Non-Compaction: Results of a Prospective Multicentric Study.

Background: Whether left ventricular non-compaction (LVNC) bears a different prognosis than dilated cardiomyopathy (DCM) is still a matter of debate. Methods: From a multicenter French prospective registry, we compared the outcomes of 98 patients with LVNC and 65 with DCM. The primary endpoint combined cardiovascular death, heart transplantation, and hospitalization for cardiovascular events. The two groups presented similar outcomes but different left ventricular ejection fractions (LVEF) (43.3% in LVNC vs. 35.95% in DCM, p = 0.001). For this reason, a subgroup analysis was performed comparing only patients with LVEF ≤ 45%, including 56 with LVNC and 49 with DCM. Results: Among patients with LVEF≤ 45%, at 5-year follow-up, the primary endpoint occurred in 33 (58.9%) among 56 patients with LVNC and 18 (36.7%) among 49 patients with DCM (p = 0.02). Hospitalization for heart failure (18 [32.14%] vs. 5 [10.20%], p = 0.035) and heart transplantation were more frequent in the LVNC than in the DCM group. The incidences of rhythmic complications (24 [42.85%] vs. 12 [24.48%], p = 0.17), embolic events, and cardiovascular death were similar between LVNC and DCM cases. Among the 42 patients with LVNC and LVEF > 45%, the primary endpoints occurred in only 4 (9.52%) patients, including 2 hospitalizations for heart failure and 3 rhythmic complications, but no embolic events. Conclusion: In this prospective cohort, patients with LVNC who have left ventricular dysfunction present a poorer prognosis than DCM patients. Heart failure events were especially more frequent, but embolic events were not. Patients with LVNC and preserved ejection fraction present very few events in 5 years.

Psychosocial Impact of Predictive Genetic Testing in Hereditary Heart Diseases: The PREDICT Study.

Predictive genetic testing (PGT) is offered to asymptomatic relatives at risk of hereditary heart disease, but the impact of result disclosure has been little studied. We evaluated the psychosocial impacts of PGT in hereditary heart disease, using self-report questionnaires (including the State-Trait Anxiety Inventory) in 517 adults, administered three times to the prospective cohort (PCo: n = 264) and once to the retrospective cohort (RCo: n = 253). The main motivations for undergoing PGT were "to remove doubt" and "for their children". The level of anxiety increased between pre-test and result appointments (p <0.0001), returned to baseline after the result (PCo), and was moderately elevated at 4.4 years (RCo). Subjects with a history of depression or with high baseline anxiety were more likely to develop anxiety after PGT result (p = 0.004 and p <0.0001, respectively), whatever it was. Unfavourable changes in professional and/or family life were observed in 12.4% (PCo) and 18.7% (RCo) of subjects. Few regrets about PGT were expressed (0.8% RCo, 2.3% PCo). Medical benefit was not the main motivation, which emphasises the role of pre/post-test counselling. When PGT was performed by expert teams, the negative impact was modest, but careful management is required in specific categories of subjects, whatever the genetic test result.

The Neural Baroreflex Pathway in Subjects With Metabolic Syndrome: A Sub-Study of the Paris Prospective Study III.

The mechanisms that link metabolic syndrome (MetS) to increased cardiovascular risk are incompletely understood. We examined whether MetS is associated with the neural baroreflex pathway (NBP) and whether any such associations are independent of blood pressure values.This study involved the cross-sectional analysis of data on 2835 subjects aged 50 to 75 years from the Paris Prospective Study 3. The prevalence of MetS was defined according to the American Heart Association/National Heart Blood and Lung Institute definition. NBP values were calculated from the fluctuation of the common carotid distension rate and heart rate using fast Fourier transformation and cross-spectral analysis.The prevalence of MetS was 20.1% in men and 10.4% in women. Compared with controls, subjects with MetS (≥3 components), and those at risk for MetS (1-2 components) had lower NBP (-5.3% and -2.3%, respectively) and higher carotid stiffness (+13.5% and +6.8%, respectively). The negative association between MetS components and NBP was confirmed, even after adjustment for age, sex, and carotid stiffness. After stratification for blood pressure (BP) levels, NBP was reduced only in MetS subjects and those at risk with high BP. The NBP was positively associated with carotid stiffness in controls and subjects at risk for MetS. This association was lost in subjects with MetS, regardless of BP levels.Subjects with MetS had reduced NBP values. The role of BP is fundamental in the reduction of NBP. The mechanisms that link carotid stiffness and NBP are inactive in subjects with MetS, independent of BP levels.