Alexandre Janin

Nuclear envelopathies: a complex LINC between nuclear envelope and pathology

Since the identification of the first disease causing mutation in the gene coding for emerin, a transmembrane protein of the inner nuclear membrane, hundreds of mutations and variants have been found in genes encoding for nuclear envelope components. These proteins can be part of the inner nuclear membrane (INM), such as emerin or SUN proteins, outer nuclear membrane (ONM), such as Nesprins, or the nuclear lamina, such as lamins A and C. However, they physically interact with each other to insure the nuclear envelope integrity and mediate the interactions of the nuclear envelope with both the genome, on the inner side, and the cytoskeleton, on the outer side. The core of this complex, called LINC (LInker of Nucleoskeleton to Cytoskeleton) is composed of KASH and SUN homology domain proteins. SUN proteins are INM proteins which interact with lamins by their N-terminal domain and with the KASH domain of nesprins located in the ONM by their C-terminal domain.Although most of these proteins are ubiquitously expressed, their mutations have been associated with a large number of clinically unrelated pathologies affecting specific tissues. Moreover, variants in SUN proteins have been found to modulate the severity of diseases induced by mutations in other LINC components or interactors. For these reasons, the diagnosis and the identification of the molecular explanation of “nuclear envelopathies” is currently challenging.The aim of this review is to summarize the human diseases caused by mutations in genes coding for INM proteins, nuclear lamina, and ONM proteins, and to discuss their potential physiopathological mechanisms that could explain the large spectrum of observed symptoms.

A fast and cost-effective molecular diagnostic tool for genetic diseases involved in sudden cardiac death

BACKGROUND Cardiomyopathies and arrhythmia syndromes are common genetic cardiac diseases that account for a significant number of sudden cardiac death (SCD) cases. METHODS NGS workflow based on a panel of 95 genes was developed on Illumina NextSeq500™ sequencer for sequencing prevalent SCD-causing genes. A cohort of 90 patients (56 genotype-positive, 27 genotype-negative and 7 new cases) was screened to evaluate this strategy in terms of sensitivity, specificity, practicability and cost. In silico analysis were performed using a pipeline based on NextGENe® software and a personalized Sophia Genetics pipeline. RESULTS Using our panel custom, 100% of targeted sequences were efficiently covered and all previously identified genetic variants were readily detected. Applied to 27 genotype-negative patients, this molecular strategy allowed the identification of pathogenic or likely pathogenic variants into 12 cases. It confirmed the involvement of HCN4 mutations in the combined bradycardia–myocardial non-compaction phenotype, and also suggested, for the first time, the involvement of PKP2, usually associated with arrhythmogenic right ventricular dysplasia, in ventricular non-compaction. CONCLUSION This NGS approach is a fast, cheap, sensitive and high-throughput mutation detection method that is ready to be deployed in clinical laboratories and would provide new insights on physiopathology of SCD, more particularly of cardiomyopathies and arrhythmia syndromes.

Truncating mutations on myofibrillar myopathies causing genes as prevalent molecular explanations on patients with Dilated Cardiomyopathy

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with high morbidity and mortality. More than 40 genes have been reported to cause DCM. To provide new insights into the pathophysiology of dilated cardiomyopathy, a next‐generation sequencing (NGS) workflow based on a panel of 48 cardiomyopathies‐causing genes was used to analyze a cohort of 222 DCM patients. Truncating variants were detected on 63 unrelated DCM cases (28.4%). Most of them were identified, as expected, on TTN (29 DCM probands), but truncating variants were also identified on myofibrillar myopathies causing genes in 17 DCM patients (7.7% of the DCM cohort): 10 variations on FLNC and 7 variations on BAG3 . This study confirms that truncating variants on myofibrillar myopathies causing genes are frequently associated with dilated cardiomyopathies and also suggest that FLNC mutations could be considered as a common cause of dilated cardiomyopathy. Molecular approaches that would allow to detect systematically truncating variants in FLNC and BAG3 into genetic testing should significantly increase test sensitivity, thereby allowing earlier diagnosis and therapeutic intervention for many patients with dilated cardiomyopathy.

Atrial fibrillation is associated with hypermethylation in human left atrium, and treatment with decitabine reduces atrial tachyarrhythmias in spontaneously hypertensive rats

&NA; Atrial fibrillation (AF) is the most common cardiac arrhythmia. As the molecular mechanisms underlying the pathology are largely unknown, this cardiac arrhythmia remains difficult to treat. To identify specific molecular actors involved in AF, we have performed a transcriptomic analysis on left atrium (LA) from patients with valvular heart disease with or without AF. We showed that 1627 genes had altered basal expression level in LA tissue of AF patients compared with the control group. The significantly enriched gene ontology biological process “anatomical structure morphogenesis” contained the highest number of genes in line with changes in structure that occur when the human heart remodels following AF development (ie, LA dilatation and interstitial fibrosis). We then focused the study on Pitx2 (paired‐like homeodomain 2), being the most altered transcription factor in LA from AF patients and from which compelling evidence have indicated that its reduced expression can be considered as a marker for the disease. In addition, its expression was inversely correlated with LA size. We demonstrated that AF is associated with Pitx2 promoter hypermethylation both in humans and arrhythmic aging spontaneously hypertensive rats. Chronic administration of a DNA methylation inhibitor (ie, 5‐Aza‐2′‐deoxycitidine) improved ECG arrhythmic profiles and superoxide dismutase activities and reduced fibrosis in the left ventricle of spontaneously hypertensive rats. Taken together, these data support the notion that AF is associated with epigenetic changes in LA and provide a proof‐of‐concept that hypomethylating agents have to be considered in the treatment of atrial arrhythmias.

HCN4 mutation as a molecular explanation on patients with bradycardia and non-compaction cardiomyopathy.

A very recent study suggested that HCN4 mutations could be associated with sinusal bradycardia and myocardial non compaction. A French family with 3 affected sisters presenting the same clinical phenotype (sinus bradycardia in combination with non compaction cardiomyopathy (NCCM)) have benefited both from a systematic cardiovascular exploration and molecular investigations. The molecular analysis, performed by NGS sequencing, led to identify only one likely-disease causing variation: p.Gly482Arg on HCN4 gene. Our results confirm the genetic evidence for the involvement of the HCN4 mutations in the combined bradycardia-NCCM phenotype and illustrates that, in front of this combined clinical phenotype, HCN4 mutations has to be suspected.

Cardiac voltage-gated sodium channel mutations associated with left atrial dysfunction and stroke in children

Aims Cardiac atrial arrhythmias are the most common type of heart rhythm disorders. Its genetic elucidation remains challenging with poor understanding of cellular and molecular processes. These arrhythmias usually affect elderly population but in rare cases, young children may also suffer from such electrical diseases. Severe complications, including stroke, are commonly age related. This study aims to identify a genetic link between electro-mechanic atrial dysfunction and stroke in children. Methods and results In two unrelated boys of 11 and 14 years with both stroke and atrial arrhythmias, the clinical phenotype was determined through a complete physical examination, electrocardiogram (ECG), Holter ECG, and computed tomography. The genetic testing was performed on a large 95 genes panel implicated in myocardial electrical imbalance, using the next generation sequencing method. The panel also includes the genes usually associated with the development of cardiomyopathies. In one child, a left atrial dilation was observed. The 2nd boy suffered from atrial standstill. Both suffered from atrial bradycardia, flutter, and fibrillation. The complete genetic testing revealed the SCN5A c.3823G>A (p.D1275N) mutation in the first family, c.1141-2A>G and c.3157G>A (p.E1053K) mutations in the second family. Conclusion Our results strengthen the association between Nav1.5 mutations and the occurrence of stroke in young patients. It emphasizes the need to look for atrial myopathy in the decision process for anticoagulation in young patients with atrial arrhythmic events.

SMAD6 overexpression leads to accelerated myogenic differentiation of LMNA mutated cells

LMNA gene encodes lamins A and C, two major components of the nuclear lamina, a network of intermediate filaments underlying the inner nuclear membrane. Most of LMNA mutations are associated with cardiac and/or skeletal muscles defects. Muscle laminopathies include Emery-Dreifuss Muscular Dystrophy, Limb-Girdle Muscular Dystrophy 1B, LMNA-related Congenital Muscular Dystrophy and Dilated Cardiomyopathy with conduction defects. To identify potential alterations in signaling pathways regulating muscle differentiation in LMNA-mutated myoblasts, we used a previously described model of conditionally immortalized murine myoblasts: H-2K cell lines. Comparing gene expression profiles in wild-type and Lmna∆8–11 H-2K myoblasts, we identified two major alterations in the BMP (Bone Morphogenetic Protein) pathway: Bmp4 downregulation and Smad6 overexpression. We demonstrated that these impairments lead to Lmna∆8–11 myoblasts premature differentiation and can be rescued by downregulating Smad6 expression. Finally, we showed that BMP4 pathway defects are also present in myoblasts from human patients carrying different heterozygous LMNA mutations.

First identification of homozygous truncating CSRP3 variants in two unrelated cases with hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease with an estimated prevalence of 1/500. More than 40 genes have been reported to cause HCM. Among them, CSRP3 is usually included on HCM gene panels used for molecular diagnosis by next-generation sequencing (NGS). To provide new insights into the pathophysiology of hypertrophic cardiomyopathy, a NGS workflow based on a panel of 48 cardiomyopathies-causing genes was analyzed on a cohort of 542 HCM patients. As expected, this molecular approach led to identify most pathogenic or likely pathogenic variants into prevalent HCM-causing genes: MYBPC3 (123/542; 22.7%), MYH7 (48/542; 8.9%), TNNT2 (12/542; 2.2%), and TNNI3 (10/542; 1.8%). Among MYBPC3 variants, 96 led to a premature stop codon (78%). More surprisingly, our molecular study led also to detect, for the first time, homozygous CSRP3 truncating variants in two unrelated HCM probands. Meta-analysis of rare previously reported CSRP3 variants on HCM probands using ACMG guidelines indicate that only one variation (p.Cys58Gly) could be considered as likely pathogen. By combining meta-analysis results and identification of two unrelated HCM patients with homozygous CSRP3 truncating variants, we suggest that the association of CSRP3 as a validated HCM-causing gene require additional studies and those CSRP3 variants could result in HCM with an autosomal recessive inheritance rather than with an autosomal dominant transmission as usually reported on HCM (OMIM 612124).