Pregnancy in women with dilated cardiomyopathy genetic variants.

INTRODUCTION AND OBJECTIVES: Limited information is available on the safety of pregnancy in patients with genetic dilated cardiomyopathy (DCM) and in carriers of DCM-causing genetic variants without the DCM phenotype. We assessed cardiac, obstetric, and fetal or neonatal outcomes in this group of patients. METHODS: We studied 48 women carrying pathogenic or likely pathogenic DCM-associated variants (30 with DCM and 18 without DCM) who had 83 pregnancies. Adverse cardiac events were defined as heart failure (HF), sustained ventricular tachycardia, ventricular assist device implantation, heart transplant, and/or maternal cardiac death during pregnancy, or labor and delivery, and up to the sixth postpartum month. RESULTS: A total of 15 patients, all with DCM (31% of the total cohort and 50% of women with DCM) experienced adverse cardiac events. Obstetric and fetal or neonatal complications were observed in 14% of pregnancies (10 in DCM patients and 2 in genetic carriers). We analyzed the 30 women who had been evaluated before their first pregnancy (12 with overt DCM and 18 without the phenotype). Five of the 12 (42%) women with DCM had adverse cardiac events despite showing NYHA class I or II before pregnancy. Most of these women had a history of cardiac events before pregnancy (80%). Among the 18 women without phenotype, 3 (17%) developed DCM toward the end of pregnancy. CONCLUSIONS: Cardiac complications during pregnancy and postpartum were common in patients with genetic DCM and were primarily related to HF. Despite apparently good tolerance of pregnancy in unaffected genetic carriers, pregnancy may act as a trigger for DCM onset in a subset of these women.

Post-mortem genetic testing in sudden cardiac death and genetic screening of relatives at risk: lessons learned from a Czech pilot multidisciplinary study.

Sudden cardiac death (SCD) might have an inherited cardiac condition background. Genetic testing supports post-mortem diagnosis and screening of relatives at risk. Our aim is to determine the feasibility of a Czech national collaboration group and to establish the clinical importance of molecular autopsy and family screening. From 2016 to 2021, we have evaluated 100 unrelated SCD cases (71.0% males, age: 33.3 (12.8) years). Genetic testing was performed by next-generation sequencing utilizing a panel of 100 genes related to inherited cardiac/aortic conditions and/or whole exome sequencing. According to autopsy, cases were divided into cardiomyopathies, sudden arrhythmic death syndrome, sudden unexplained death syndrome, and sudden aortic death. We identified pathogenic/likely pathogenic variants following ACMG/AMP recommendations in 22/100 (22.0%) of cases. Since poor DNA quality, we have performed indirect DNA testing in affected relatives or in healthy parents reaching a diagnostic genetic yield of 11/24 (45.8%) and 1/10 (10.0%), respectively. Cardiological and genetic screening disclose 83/301 (27.6%) relatives at risk of SCD. Genetic testing in affected relatives as starting material leads to a high diagnostic yield offering a valuable alternative when suitable material is not available. This is the first multidisciplinary/multicenter molecular autopsy study in the Czech Republic which supports the establishment of this type of diagnostic tests. A central coordinator and proper communication among centers are crucial for the success of a collaboration at a national level.

Isolated aneurysmal disease as an underestimated finding in individuals with JAG1 pathogenic variants.

Pathogenic variants in JAG1 are known to cause Alagille syndrome (ALGS), a disorder that primarily affects the liver, lung, kidney, and skeleton. Whereas cardiac symptoms are also frequently observed in ALGS, thoracic aortic aneurysms have only been reported sporadically in postmortem autopsies. We here report two families with segregating JAG1 variants that present with isolated aneurysmal disease, as well as the first histological evaluation of aortic aneurysm tissue of a JAG1 variant carrier. Our observations shed more light on the pathomechanisms behind aneurysm formation in JAG1 variant harboring individuals and underline the importance of cardiovascular imaging in the clinical follow-up of such individuals.

Novel LOX Variants in Five Families with Aortic/Arterial Aneurysm and Dissection with Variable Connective Tissue Findings.

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.

Pigmentary retinopathy can indicate the presence of pathogenic LAMP2 variants even in somatic mosaic carriers with no additional signs of Danon disease.

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.

Danon disease is an underdiagnosed cause of advanced heart failure in young female patients: a LAMP2 flow cytometric study.

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.

Desminopathy: Novel Desmin Variants, a New Cardiac Phenotype, and Further Evidence for Secondary Mitochondrial Dysfunction.

Background: The pleomorphic clinical presentation makes the diagnosis of desminopathy difficult. We aimed to describe the prevalence, phenotypic expression, and mitochondrial function of individuals with putative disease-causing desmin (DES) variants identified in patients with an unexplained etiology of cardiomyopathy. Methods: A total of 327 Czech patients underwent whole exome sequencing and detailed phenotyping in probands harboring DES variants. Results: Rare, conserved, and possibly pathogenic DES variants were identified in six (1.8%) probands. Two DES variants previously classified as variants of uncertain significance (p.(K43E), p.(S57L)), one novel DES variant (p.(A210D)), and two known pathogenic DES variants (p.(R406W), p.(R454W)) were associated with characteristic desmin-immunoreactive aggregates in myocardial and/or skeletal biopsy samples. The individual with the novel DES variant p.(Q364H) had a decreased myocardial expression of desmin with absent desmin aggregates in myocardial/skeletal muscle biopsy and presented with familial left ventricular non-compaction cardiomyopathy (LVNC), a relatively novel phenotype associated with desminopathy. An assessment of the mitochondrial function in four probands heterozygous for a disease-causing DES variant confirmed a decreased metabolic capacity of mitochondrial respiratory chain complexes in myocardial/skeletal muscle specimens, which was in case of myocardial succinate respiration more profound than in other cardiomyopathies. Conclusions: The presence of desminopathy should also be considered in individuals with LVNC, and in the differential diagnosis of mitochondrial diseases.

Alu-mediated Xq24 deletion encompassing CUL4B, LAMP2, ATP1B4, TMEM255A, and ZBTB33 genes causes Danon disease in a female patient.

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.

LAMP2 exon-copy number variations in Danon disease heterozygote female probands: Infrequent or underdetected?

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.

Corrigendum: Candidate Gene Resequencing in a Large Bicuspid Aortic Valve-Associated Thoracic Aortic Aneurysm Cohort: SMAD6 as an Important Contributor.

[This corrects the article on p. 400 in vol. 8, PMID: 28659821.].

Candidate Gene Resequencing in a Large Bicuspid Aortic Valve-Associated Thoracic Aortic Aneurysm Cohort: SMAD6 as an Important Contributor.

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.

Distribution of CFTR mutations in the Czech population: positive impact of integrated clinical and laboratory expertise, detection of novel/de novo alleles and relevance for related/derived populations.

BACKGROUND: This two decade long study presents a comprehensive overview of the CFTR mutation distribution in a representative cohort of 600 Czech CF patients derived from all regions of the Czech Republic. METHODS: We examined the most common CF-causing mutations using the Elucigene CF-EU2v1™ assay, followed by MLPA, mutation scanning and/or sequencing of the entire CFTR coding region and splice site junctions. RESULTS: We identified 99.5% of all mutations (1194/1200 CFTR alleles) in the Czech CF population. Altogether 91 different CFTR mutations, of which 20 were novel, were detected. One case of de novo mutation and a novel polymorphism was revealed. CONCLUSION: The commercial assay achieved 90.7%, the MLPA added 1.0% and sequencing increased the detection rate by 7.8%. These comprehensive data provide a basis for the improvement of CF DNA diagnostics and/or newborn screening in our country. In addition, they are relevant to related Central European populations with lower mutation detection rates, as well as to the sizeable North American "Bohemian diaspora".

High positive predictive value of PAPP-A for acute coronary syndrome diagnosis in heparin-naïve patients.

Pregnancy-associated plasma protein-A (PAPP-A) was studied as a marker of acute coronary syndrome (ACS). It has been shown that its levels are increased by heparin administration. Therefore, the aim of our study was to ascertain the diagnostic significance of PAPP-A in heparin-naïve patients and compare it with (TnI). We prospectively studied 67 heparin-naïve patients with acute chest pain. The patients were independently grouped according to the presence or absence of ACS. PAPP-A levels were significantly increased in ACS patients (8.6 vs. 7.3 mIU/l; P = 0.006) with high positive (95.7%) and lower negative predictive values (47.7%). In multivariate analysis, its levels were strongly predictive of a final diagnosis of ACS (OR 41.8; 95th CI 2.64-662.6; P = 0.008). The diagnostic significance of PAPP-A was not higher than TnI even within 6 h after the onset of chest pain [the area under the ROC curve (AUC) was 0.69 for PAPP-A and 0.91 for TnI, respectively; P = 0.08]. We observed no difference in the AUC in NSTE-ACS patients (0.73 for PAPP-A and 0.79 for TnI (P = 0.5)). PAPP-A levels were an independent predictor of ACS diagnosis in heparin-naïve patients. Its diagnostic significance was not higher than TnI even within a short period after the onset of chest pain. In troponin-negative NSTE-ACS patients, PAPP-A helped make the correct final diagnosis.

Influence of concomitant heparin administration on pregnancy-associated plasma protein-A levels in acute coronary syndrome with ST segment elevation.

INTRODUCTION: The time course of pregnancy-associated plasma protein-A (PAPP-A) levels was studied at admission, immediately after percutaneous coronary intervention (PCI) and 1, 2, 4, 6, 12, 24 and 48 h after PCI in acute coronary syndrome with ST segment elevation (ACS-STE) to determine the impact of PCI, concomitant clinical complications and heparin administration. MATERIAL AND METHODS: Pregnancy-associated plasma protein-A serum levels, examined by the Kryptor(TM) system, were studied in 30 heparinized PCI ACS-STE patients, in 10 elective PCIs and 12 coronary angiographies with heparin, and in 5 patients with normal coronary angiogram without heparin. RESULTS: Heparin caused a high PAPP-A increase in ACS-STE patients, in all patients with heparin without ACS and angiographic signs of significant atherosclerosis. This increase was directly associated with heparin dosage and activated clotting time (ACT) (r = 0.71, p = 0.0001) and inversely with the interval between heparin applications and time of serum sampling. It was followed by a rapid decrease within 1 to 2 h and return to normal levels in 10 to 12 h. In ACS-STE patients the decrease was significantly slower than in heparinized elective PCI and angiography patients. The PAPP-A increase was not significantly dependent on the length of PCI. Persistent increase after 24 h was associated in 4/7 patients with concomitant clinical complications. CONCLUSIONS: The diagnostic validity of PAPP-A can be verified only within the 1(st) h after clinical onset of ACS before heparin administration, the prognostic value in heparinized patients not earlier than 12 h after the last heparin application, if ACT is normal and serious clinical concomitant complications are eliminated.

Fine mapping of autosomal dominant nonsyndromic hearing impairment DFNA21 to chromosome 6p24.1-22.3.

Previously, the DFNA21 locus was positioned telomeric to the DFNA13 locus based on testing of candidate loci. One family member in this region did not carry the at risk haplotype, although he had the same nonspecific midfrequency hearing impairment as other affected family members. Hence, we performed a whole genome linkage scan excluding other regions of the genome and confirming the localization of DFNA21 to 6p22.3-24.1. The DFNA21 interval was determined to span 12.4 Mb (approximately 22 cM) and is delimited on the telomeric side by BV097155 and on the centromeric side by D6S1691. A maximum lod score of 3.51 (theta = 0.066), was calculated for marker D6S1721. The DFNA21 region does not overlap the adjacent DFNA31 and DFNA13 loci and contains 31 known genes. The coding regions and exon-intron boundaries of four candidate genes, SOX4, MYLIP, CAP2, and RPEL1, were sequenced, but no mutations were identified.

QF-PCR examination of parental and meiotic origin of trisomy 21 in Central and Eastern Europe.

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.

Homozygosity mapping of a second gene locus for hereditary combined deficiency of vitamin K-dependent clotting factors to the centromeric region of chromosome 16.

Familial multiple coagulation factor deficiency (FMFD) of factors II, VII, IX, X, protein C, and protein S is a very rare bleeding disorder with autosomal recessive inheritance. The phenotypic presentation is variable with respect to the residual activities of the affected proteins, its response to oral administration of vitamin K, and to the involvement of skeletal abnormalities. The disease may result either from a defective resorption/transport of vitamin K to the liver, or from a mutation in one of the genes encoding gamma-carboxylase or other proteins of the vitamin K cycle. We have recently presented clinical details of a Lebanese family and a German family with 10 and 4 individuals, respectively, where we proposed autosomal recessive inheritance of the FMFD phenotype. Biochemical investigations of vitamin K components in patients' serum showed a significantly increased level of vitamin K epoxide, thus suggesting a defect in one of the subunits of the vitamin K 2,3-epoxide reductase (VKOR) complex. We now have performed a genome-wide linkage analysis and found significant linkage of FMFD to chromosome 16. A total maximum 2-point LOD score of 3.4 at theta = 0 was obtained in the interval between markers D16S3131 on 16p12 and D16S419 on 16q21. In both families, patients were autozygous for 26 and 28 markers, respectively, in an interval of 3 centimorgans (cM). Assuming that FMFD and warfarin resistance are allelic, conserved synteny between human and mouse linkage groups would restrict the candidate gene interval to the centromeric region of the short arm of chromosome 16.

Premature chromosome condensation in humans associated with microcephaly and mental retardation: a novel autosomal recessive condition.

We report a novel autosomal recessive disorder characterized by premature chromosome condensation in the early G2 phase. It was observed in two siblings, from consanguineous parents, affected with microcephaly, growth retardation, and severe mental retardation. Chromosome analysis showed a high frequency of prophase-like cells (>10%) in lymphocytes, fibroblasts, and lymphoblast cell lines with an otherwise normal karyotype. (3)H-thymidine-pulse labeling and autoradiography showed that, 2 h after the pulse, 28%-35% of the prophases were labeled, compared with 9%-11% in healthy control subjects, indicating that the phenomenon is due to premature chromosome condensation. Flow cytometry studies demonstrate that the entire cell cycle is not prolonged, compared with that in healthy control subjects, and compartment sizes did not differ from those in healthy control subjects. No increased reaction of the cells to X-irradiation or treatments with the clastogens bleomycin and mitomycin C was observed, in contrast to results in the cell-cycle mutants ataxia telangiectasia and Fanconi anemia. The rates of sister chromatid exchanges and the mitotic nondisjunction rates were inconspicuous. Premature entry of cells into mitosis suggests that a gene involved in cell-cycle regulation is mutated in these siblings.

Association of human aging with a functional variant of klotho.

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.