Celeghin, Rudy (2019) Genomics in Arrhythmogenic Cardiomyopathy: exploring the complexity. [Ph.D. thesis]
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Background Arrhythmogenic cardiomyopathy (AC) is a rare heart muscle disease characterized by fibrofatty myocardial replacement, prominent impairment of ventricular systolic function and arrhythmias. AC phenotypic spectrum was revealed wider than previously thought thanks to genotype–phenotype correlation. Combining multiple sources of clinical information, such as genetic, electrocardiographic, arrhythmic, morphofunctional, and histopathologic findings resulted the best approach to untangle the complexity of this disease.
The aim of our study was to assess AC genetic heterogeneity, applying appropriate genetic screening and performing a multiparametric genotype-phenotype correlation taking into account AC variants classification. Materials and Methods A total of 224 consecutive patients, with a clinical (n.192) or post mortem (n.32) diagnosis of AC, underwent genetic screening by using a 174 cardiac-related genes panel (Trusight Cardio, Illumina). The prevalence of Copy Number Variations (CNVs) and newly AC-associated genes such as FLNC and CDH2, were investigated. Detailed clinical data were obtained on 12-lead elettrocardiography, echocardiography, and cardiac magnetic resonance with the purpose of performing a multiparametric genotype-phenotype correlation. TTN variants were evaluated separately, due the magnitude of the gene, as well as rare variants in AC-unrelated genes. Finally, WES was carried out on 10 AC genotype negative patients in order to identify new candidate genes involved in the disease pathogenesis. Results We identified 95 different rare genetic variants in 97 (43%) of the 224 index cases. Of them, 79 variants were found in 5 major desmosomal genes (83%), whereas 16 in AC-related non desmosomal genes (17%). American College of Medical Genetics (ACMG)-based variant classification made evident that half of desmosomal variants (39/79, 49%) were classified as pathogenic/likely pathogenic and were predominantly radical (32/39, 78%). Comprehensive sequencing, including newly AC associated genes (FLNC and CDH2) and CNVs analysis, led to the identification of the genetic cause in 10 more patients increasing the overall yield of genetic screening from 43% to 48%. No pathogenic variants were identified in 117 (52%) patients.
Overall we re-evaluated based on the current International Task Force Criteria (ITC) the clinical phenotype of 188 out of 224 AC (84%) index cases. Of these, 94 index cases received a definite AC diagnosis at the outpatient clinic, 16 were heart-transplanted (HTx) patients and 32 sudden death (SD) victims. The remaining 18 were borderline and 28 were possibly affected by AC. 78 of the 142 (55%) definite index cases carried at least a rare variant in AC related genes whereas, only 13 of the 46 (28%) borderline/possible index cases were genotype positive.
Genotype analysis focusing on ventricular involvement highlighted that patients with Left Dominant variant (LDAC) were significantly less positive for desmosomal variants (11/42, 26%) compared to the “classic” AC cases (75/182, 41%)(p-value 0.0065). More in deep, 19 of the 42 (45%) LDAC patients were SD victims, of whom only 4 cases (21%) were genotype positive for desmosomal rare variants.
Based on previous transcriptome studies from our laboratory we identified rare variants in LGALS3. Specifically, sequencing of 10 index cases through WES and 140 by direct sequencing, led to the identification of 5 LGALS3 rare nucleotide variants in 7 probands (4%, 5 males, mean age 39±11 years). Of note, two missense variants occurred in the protein carbohydrate recognition domain (CRD) conferring the loss of its binding site.
Comprehensive genetic analysis revealed a genetic cause in nearly half (48%) of AC patients, of which only half could be classified as P/LP. A proper phenotypic characterization increased variant finding likelihood in definite AC patients (55%). Nevertheless, half of AC patients still missed a genetic cause. Specifically, genetic testing achieved to identify a causative variant in only ~25% of LDAC cases. Finally a new candidate gene was identified in 4% of AC cases, supporting the fact that other genetic factors might be involved in disease pathogenesis. Most of identified genetic variants were variants of unknown significance (VUS), highlighting that cascade genetic screening remains mandatory to understand their significance in disease pathogenesis.
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