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Abstract Aims Recent‐onset dilated cardiomyopathy (RODCM) is characterized by heterogeneous aetiology and diverse clinical outcomes, with scarce data on genotype–phenotype correlates. Our aim was to correlate individual RODCM genotypes with left ventricular reverse remodelling (LVRR) and clinical outcomes. Methods and results In this prospective study, a total of 386 Czech RODCM patients with symptom duration ≤6 months underwent genetic counselling and whole‐exome sequencing (WES). The presence of pathogenic (class 5) or likely pathogenic (class 4) variants in a set of 72 cardiomyopathy‐related genes was correlated with the occurrence of all‐cause death, heart transplantation, or implantation of a ventricular assist device (primary outcome) and/or ventricular arrhythmia event (secondary outcome). LVRR was defined as an improvement of left ventricular ejection fraction to >50% or ≥10% absolute increase, with a left ventricular end‐diastolic diameter ≤33 mm/m2 or ≥10% relative decrease. Median follow‐up was 41 months. RODCM was familial in 98 (25%) individuals. Class 4–5 variants of interest (VOIs) were identified in 125 (32%) cases, with 69 (18%) having a single titin‐truncating variant (TTNtv) and 56 (14%) having non‐titin (non‐TTN) VOIs. The presence of class 4–5 non‐TTN VOIs, but not of TTNtv, heralded a lower probability of 12‐month LVRR and proved to be an independent baseline predictor both of the primary and the secondary outcome. The negative result of genetic testing was a strong protective baseline variable against occurrence of life‐threatening ventricular arrhythmias. Detection of class 4–5 VOIs in genes coding nuclear envelope proteins was another independent predictor of both study outcomes at baseline and also of life‐threatening ventricular arrhythmias after 12 months. Class 4–5 VOIs of genes coding cytoskeleton were associated with an increased risk of life‐threatening ventricular arrhythmias after baseline assessment. A positive family history of dilated cardiomyopathy alone only related to a lower probability of LVRR at 12 months and at the final follow‐up. Conclusions RODCM patients harbouring class 4–5 non‐TTN VOIs are at higher risk of progressive heart failure and life‐threatening ventricular arrhythmias. Genotyping may improve their early risk stratification at baseline assessment.

Aims: Recent‐onset dilated cardiomyopathy (RODCM) is characterized by heterogeneous aetiology and diverse clinical outcomes, with scarce data on genotype–phenotype correlates. Our aim was to correlate individual RODCM genotypes with left ventricular reverse remodelling (LVRR) and clinical outcomes. Methods and results: In this prospective study, a total of 386 Czech RODCM patients with symptom duration ≤6 months underwent genetic counselling and whole‐exome sequencing (WES). The presence of pathogenic (class 5) or likely pathogenic (class 4) variants in a set of 72 cardiomyopathy‐related genes was correlated with the occurrence of all‐cause death, heart transplantation, or implantation of a ventricular assist device (primary outcome) and/or ventricular arrhythmia event (secondary outcome). LVRR was defined as an improvement of left ventricular ejection fraction to >50% or ≥10% absolute increase, with a left ventricular end‐diastolic diameter ≤33 mm/m2 or ≥10% relative decrease. Median follow‐up was 41 months. RODCM was familial in 98 (25%) individuals. Class 4–5 variants of interest (VOIs) were identified in 125 (32%) cases, with 69 (18%) having a single titin‐truncating variant (TTNtv) and 56 (14%) having non‐titin (non‐TTN) VOIs. The presence of class 4–5 non‐TTN VOIs, but not of TTNtv, heralded a lower probability of 12‐month LVRR and proved to be an independent baseline predictor both of the primary and the secondary outcome. The negative result of genetic testing was a strong protective baseline variable against occurrence of life‐threatening ventricular arrhythmias. Detection of class 4–5 VOIs in genes coding nuclear envelope proteins was another independent predictor of both study outcomes at baseline and also of life‐threatening ventricular arrhythmias after 12 months. Class 4–5 VOIs of genes coding cytoskeleton were associated with an increased risk of life‐threatening ventricular arrhythmias after baseline assessment. A positive family history of dilated cardiomyopathy alone only related to a lower probability of LVRR at 12 months and at the final follow‐up. Conclusions: RODCM patients harbouring class 4–5 non‐TTN VOIs are at higher risk of progressive heart failure and life‐threatening ventricular arrhythmias. Genotyping may improve their early risk stratification at baseline assessment. [ABSTRACT FROM AUTHOR]

Abstract Aims Danon disease (DD) is a rare X‐linked disorder caused by mutations in the lysosomal‐associated membrane protein type 2 gene (LAMP2). DD is difficult to distinguish from other causes of dilated or hypertrophic cardiomyopathy (HCM) in female patients. As DD female patients regularly progress into advanced heart failure (AHF) aged 20–40 years, their early identification is critical to improve patient survival and facilitate genetic counselling. In this study, we evaluated the prevalence of DD among female patients with non‐ischemic cardiomyopathy, who reached AHF and were younger than 40 years. Methods and results The study cohort comprised 60 female patients: 47 (78%) heart transplant recipients, 2 (3%) patients treated with ventricular assist device, and 11 (18%) patients undergoing pre‐transplant assessment. Aetiology of the cardiomyopathy was known in 15 patients (including two DD patients). LAMP2 expression in peripheral white blood cells (WBC) was tested by flow cytometry (FC) in the remaining 45 female patients. Whole exome sequencing was used as an alternative independent testing method to FC. Five additional female DD patients (two with different novel LAMP2 mutations) were identified by FC. The total prevalence of DD in this cohort was 12%. HCM phenotype (57% vs. 9%, *P = 0.022) and delta waves identified by electrocardiography (43% vs. 0%, **P = 0.002) were significantly more frequent in DD female patients. Conclusions Danon disease is an underdiagnosed cause of AHF in young female patients. LAMP2 expression testing in peripheral WBCs by FC can be used as an effective screening/diagnostic tool to identify DD in this patient population.

Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6, and MAT2A. Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3) or in syndromic (e.g., TGFBR1/2, TGFB2/3) or non-syndromic (e.g., ACTA2) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort (n = 441; BAV in addition to an aortic root or ascendens diameter ≥ 4.0 cm in adults, or a Z-score ≥ 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation (n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 (p = 0.002), with 2.5% (n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype.

Mice deficient in Klotho gene expression exhibit a syndrome resembling premature human aging. To determine whether variation in the human KLOTHO locus contributes to survival, we applied two newly characterized polymorphic microsatellite markers flanking the gene in a population-based association study. In a cohort chosen for its homogeneity, Bohemian Czechs, we demonstrated significant differences in selected marker allele frequencies between newborn and elderly individuals (P < 0.05). These results precipitated a search for functional variants of klotho. We identified an allele, termed KL-VS, containing six sequence variants in complete linkage disequilibrium, two of which result in amino acid substitutions F352V and C370S. Homozygous elderly individuals were underrepresented in three distinct populations: Bohemian Czechs, Baltimore Caucasians, and Baltimore African-Americans [combined odds ratio (OR) = 2.59, P < 0.0023]. In a transient transfection assay, secreted levels of klotho harboring V352 are reduced 6-fold, whereas extracellular levels of the S370 form are increased 2.9-fold. The V352/S370 double mutant exhibits an intermediate phenotype (1.6-fold increase), providing a rare example of intragenic complementation in cis by human single nucleotide polymorphisms. The remarkable conservation of F352 among homologous proteins suggests that it is functionally important. The corresponding substitution, F289V, in the closest human klotho paralog with a known substrate, cBGL1, completely eliminates its ability to cleave p-nitrophenyl-[beta]-D-glucoside. These results suggest that the KL-VS allele influences the trafficking and catalytic activity of klotho, and that variation in klotho function contributes to heterogeneity in the onset and severity of human age-related phenotypes.

Correlation between genotype and phenotype has been examined in 14 families with autosomal recessive lamellar ichthyosis. The results show no consistent correlation between genetic and clinical classifications, suggesting that existing clinical criteria do not discriminate between the different molecular forms of the disease. Three loci have been associated with lamellar ichthyosis. In families with mutations on the TGM1 gene, seven different missense mutations and one splice mutation have been found.

Purpose: Danon disease (DD) is a rare X‐linked disorder caused by pathogenic variants in LAMP2. DD primarily manifests as a severe cardiomyopathy. An early diagnosis is crucial for patient survival. The aim of the study was to determine the usefulness of ocular examination for identification of DD. Methods: Detailed ocular examination in 10 patients with DD (3 males, 7 females) and a 45‐year‐old asymptomatic female somatic mosaic carrier of a LAMP2 disease‐causing variant. Results: All patients with manifest cardiomyopathy had pigmentary retinopathy with altered autofluorescence and diffuse visual field loss. Best corrected visual acuity (BCVA) was decreased (<0.63) in 8 (40%) out of 20 eyes. The severity of retinal pathology increased with age, resulting in marked cone‐rod involvement overtime. Spectral‐domain optical coherence tomography in younger patients revealed focal loss of photoreceptors, disruption and deposition at the retinal pigment epithelium/Bruch's membrane layer (corresponding to areas of marked increased autofluorescence), and hyperreflective foci in the outer nuclear layer. Cystoid macular oedema was seen in one eye. In the asymptomatic female with somatic mosaicism, the BCVA was 1.0 bilaterally. An abnormal autofluorescence pattern in the left eye was present; while full‐field electroretinography was normal. Conclusions: Detailed ocular examination may represent a sensitive and quick screening tool for the identification of carriers of LAMP2 pathogenic variants, even in somatic mosaicism. Hence, further investigation should be undertaken in all patients with pigmentary retinal dystrophy as it may be a sign of a life‐threatening disease. [ABSTRACT FROM AUTHOR]

Aims: Danon disease (DD) is a rare X‐linked disorder caused by mutations in the lysosomal‐associated membrane protein type 2 gene (LAMP2). DD is difficult to distinguish from other causes of dilated or hypertrophic cardiomyopathy (HCM) in female patients. As DD female patients regularly progress into advanced heart failure (AHF) aged 20–40 years, their early identification is critical to improve patient survival and facilitate genetic counselling. In this study, we evaluated the prevalence of DD among female patients with non‐ischemic cardiomyopathy, who reached AHF and were younger than 40 years. Methods and results: The study cohort comprised 60 female patients: 47 (78%) heart transplant recipients, 2 (3%) patients treated with ventricular assist device, and 11 (18%) patients undergoing pre‐transplant assessment. Aetiology of the cardiomyopathy was known in 15 patients (including two DD patients). LAMP2 expression in peripheral white blood cells (WBC) was tested by flow cytometry (FC) in the remaining 45 female patients. Whole exome sequencing was used as an alternative independent testing method to FC. Five additional female DD patients (two with different novel LAMP2 mutations) were identified by FC. The total prevalence of DD in this cohort was 12%. HCM phenotype (57% vs. 9%, *P = 0.022) and delta waves identified by electrocardiography (43% vs. 0%, **P = 0.002) were significantly more frequent in DD female patients. Conclusions: Danon disease is an underdiagnosed cause of AHF in young female patients. LAMP2 expression testing in peripheral WBCs by FC can be used as an effective screening/diagnostic tool to identify DD in this patient population. [ABSTRACT FROM AUTHOR]

Cullin 4B (CUL4B), lysosomal‐associated membrane protein Type 2 (LAMP2), ATP1B4, TMEM255A, and ZBTB33 are neighboring genes on Xq24. Mutations in CUL4B result in Cabezas syndrome (CS). Male CS patients present with dysmorphic, neuropsychiatric, genitourinary, and endocrine abnormalities. Heterozygous CS females are clinically asymptomatic. LAMP2 mutations cause Danon disease (DD). Cardiomyopathy is a dominant feature of DD present in both males and heterozygous females. No monogenic phenotypes have been associated with mutations in ATP1B4, TMEM255A, and ZBTB33 genes. To facilitate diagnostics and counseling in CS and DD families, we present a female DD patient with a de novo Alu‐mediated Xq24 rearrangement causing a deletion encompassing CUL4B, LAMP2, and also the other three neighboring genes. Typical to females heterozygous for CUL4B mutations, the patient was CS asymptomatic, however, presented with extremely skewed X‐chromosome inactivation (XCI) ratios in peripheral white blood cells. As a result of the likely selection against CUL4B deficient clones, only minimal populations (~3%) of LAMP2 deficient leukocytes were identified by flow cytometry. On the contrary, myocardial LAMP2 protein expression suggested random XCI. We demonstrate that contiguous CUL4B and LAMP2 loss‐of‐function copy number variations occur and speculate that male patients carrying similar defects could present with features of both CS and DD. [ABSTRACT FROM AUTHOR]

Danon disease (DD) is an X‐linked disorder caused by mutations in the lysosomal‐associated membrane protein 2 (LAMP2) gene (Xq24). DD is characterized by cognitive deficit, myopathy, and cardiomyopathy in male patients. The phenotype is variable and mitigated in females. The timely identification of de‐novo LAMP2 mutated family members, many of whom are heterozygous females, remains critical for their treatment and family counseling. DD laboratory testing builds on minimally invasive quantification of the LAMP2 protein in white blood cells and characterization of the specific mutation. This integrative approach is particularly helpful when assessing suspect female heterozygotes. LAMP2 exon‐copy number variations (eCNVs) were so far reported only in X‐hemizygous male DD probands. In heterozygous female DD probands, the wild‐type allele may hamper the identification of an eCNV even if it results in the complete abolition of LAMP2 transcription and/or translation. To document the likely underappreciated rate of occurrence and point out numerous potential pitfalls of detection of the LAMP2 eCNVs, we present the first two DD heterozygote female probands who harbor novel multi‐exon LAMP2 deletions. Critical for counseling and recurrence prediction, we also highlight the need to search for somatic‐germinal mosaicism in DD families. [ABSTRACT FROM AUTHOR]

Thoracic aortic aneurysm and dissection (TAAD) is a major cause of cardiovascular morbidity and mortality. Loss-of-function variants in LOX, encoding the extracellular matrix crosslinking enzyme lysyl oxidase, have been reported to cause familial TAAD. Using a next-generation TAAD gene panel, we identified five additional probands carrying LOX variants, including two missense variants affecting highly conserved amino acids in the LOX catalytic domain and three truncating variants. Connective tissue manifestations are apparent in a substantial fraction of the variant carriers. Some LOX variant carriers presented with TAAD early in life, while others had normal aortic diameters at an advanced age. Finally, we identified the first patient with spontaneous coronary artery dissection carrying a LOX variant. In conclusion, our data demonstrate that loss-of-function LOX variants cause a spectrum of aortic and arterial aneurysmal disease, often combined with connective tissue findings. [ABSTRACT FROM AUTHOR]

Studies the detection of a cystic fibrosis modifier locus for meconium ileus on human chromosome 19q13. Identification of polymorphic miscrosatellite markers; Genotypes of the sibpairs at the locus; Evaluation of clinical phenotypes; Number of alleles identical by descent at each marker loci; Distribution of sibpairs in relation to three-marker haplotype sharing.

Bicuspid aortic valve (BAV) is the most common congenital heart defect. Although many BAV patients remain asymptomatic, at least 20% develop thoracic aortic aneurysm (TAA). Historically, BAV-related TAA was considered as a hemodynamic consequence of the valve defect. Multiple lines of evidence currently suggest that genetic determinants contribute to the pathogenesis of both BAV and TAA in affected individuals. Despite high heritability, only very few genes have been linked to BAV or BAV/TAA, such as NOTCH1, SMAD6 , and MAT2A . Moreover, they only explain a minority of patients. Other candidate genes have been suggested based on the presence of BAV in knockout mouse models (e.g., GATA5, NOS3 ) or in syndromic (e.g., TGFBR1/2, TGFB2/3 ) or non-syndromic (e.g., ACTA2 ) TAA forms. We hypothesized that rare genetic variants in these genes may be enriched in patients presenting with both BAV and TAA. We performed targeted resequencing of 22 candidate genes using Haloplex target enrichment in a strictly defined BAV/TAA cohort ( n = 441; BAV in addition to an aortic root or ascendens diameter ≥ 4.0 cm in adults, or a Z-score ≥ 3 in children) and in a collection of healthy controls with normal echocardiographic evaluation ( n = 183). After additional burden analysis against the Exome Aggregation Consortium database, the strongest candidate susceptibility gene was SMAD6 ( p = 0.002), with 2.5% ( n = 11) of BAV/TAA patients harboring causal variants, including two nonsense, one in-frame deletion and two frameshift mutations. All six missense mutations were located in the functionally important MH1 and MH2 domains. In conclusion, we report a significant contribution of SMAD6 mutations to the etiology of the BAV/TAA phenotype.

Study of parental/meiotic origin of free trisomy 21 in nuclear families from Russia (70 cases), Ukraine (32 cases), and 22 from Germany revealed maternal nondisjunction in 77.3% (Germany), 93.8% (Ukraine), and 91.4% (Russia), paternal origin in 13.6%, 6.2%, and 8.6%, respectively. Maternal meiosis I errors were found in 84.4% (Ukraine), 77.1% (Russia), paternal origin in 3.1% (Ukraine), 2.9% (Russia). Maternal meiosis II errors occurred in 9.4% and 14.3% and paternal in 3.1% and 5.7% in Ukraine and Russia, respectively. No significant differences were found in maternal/paternal origin among Ukraine, Russia, Germany, and published data from other European regions.