Patients with hypertrophic obstructive cardiomyopathy after alcohol septal ablation have favorable long-term outcome irrespective of their genetic background.

BACKGROUND: The genetic background of patients with hypertrophic cardiomyopathy (HCM) treated with alcohol septal ablation (ASA) and its relationship to the outcomes are not known. We aimed to investigate whether the outcome of genotype positive (G+) patients differs from genotype negative (G-) patients treated with ASA. METHODS: We included 129 HCM patients (mean age 54±13 years) treated with ASA in a tertiary cardiovascular center and performed next generation sequencing (NGS) based genomic testing. All patients were followed-up three months after the procedure and yearly thereafter. RESULTS: A total of 30 (23%) HCM patients were G+ patients. At the 3-months follow-up, both groups of patients had similar left ventricular outflow tract PG (16.9±15.7 mmHg in G+ vs. 16.3±18.8 mmHg in G-, P=0.73) and symptoms (follow-up NYHA class 1.40±0.62 vs. 1.37±0.53, P=0.99, follow-up CCS class 0.23±0.52 vs. 0.36±0.65, P=0.36). The independent predictors of all-cause mortality were baseline interventricular septum (IVS) thickness (HR 1.12, 95% CI: 1.00-1.26, P=0.049) and age at the time of ASA (HR 1.11, 95% CI: 1.06-1.17, P<0.01). The adjusted all-cause mortality rate did not differ significantly between G+ and G- patients (P=0.52). The adjusted combined mortality event rate did not differ between both groups (P=0.78). CONCLUSIONS: Despite more severe phenotype in G+ HCM patients, ASA is an equally effective treatment for LVOTO in G+ patients as it is for treating LVOTO in G- patients. The long-term outcome after ASA is similar in G+ and G- patients.

The utility of the Mayo Score for predicting the yield of genetic testing in patients with hypertrophic cardiomyopathy.

INTRODUCTION: The yield of genetic testing in hypertrophic cardiomyopathy (HCM) is variable. The Mayo HCM Genotype Predictor score (Mayo Score) provides the pre-test probability of a positive HCM genetic test. In the original cohort of Mayo Score patients, only 9 HCM-associated myofilament genes were evaluated. The aim of this study was to validate the Mayo Score in the national HCM cohort and assess the yield of genetic testing using next generation sequencing (NGS) evaluating up to 229 genes. MATERIAL AND METHODS: We included 336 consecutive unrelated HCM patients (41% women, mean age: 53 ±15 years). We performed NGS-based genomic testing with classification of identified variants according to American College of Medical Genetics and Genomics guidelines. NGS findings were compared with the Mayo Score (ranging from -1 to 5) based on clinical and echocardiographic variables. RESULTS: We identified 72 variants classified as pathogenic or likely pathogenic in 70 (21%) HCM patients. One patient with the highest Mayo Score of 5 had a pathogenic mutation (100% yield). Patients with a Mayo Score of 4 had a pathogenic mutation in 71% of cases. Patients with a Mayo Score of 3 or 2 had a pathogenic mutation in 50 and 35% of cases, respectively. The yield of genetic testing in patients with a Mayo Score of -1 to 1 was low (6-21%). CONCLUSIONS: The overall yield of genetic testing using NGS evaluating up to 229 genes was low. The yield of genetic testing was consistently predicted with Mayo Score values.

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.

Under the mask of Kabuki syndrome: Elucidation of genetic-and phenotypic heterogeneity in patients with Kabuki-like phenotype.

Kabuki syndrome is mainly caused by dominant de-novo pathogenic variants in the KMT2D and KDM6A genes. The clinical features of this syndrome are highly variable, making the diagnosis of Kabuki-like phenotypes difficult, even for experienced clinical geneticists. Herein we present molecular genetic findings of causal genetic variation using array comparative genome hybridization and a Mendeliome analysis, utilizing targeted exome analysis focusing on regions harboring rare disease-causing variants in Kabuki-like patients which remained KMT2D/KDM6A-negative. The aCGH analysis revealed a pathogenic CNV in the 14q11.2 region, while targeted exome sequencing revealed pathogenic variants in genes associated with intellectual disability (HUWE1, GRIN1), including a gene coding for mandibulofacial dysostosis with microcephaly (EFTUD2). Lower values of the MLL2-Kabuki phenotypic score are indicative of Kabuki-like phenotype (rather than true Kabuki syndrome), where aCGH and Mendeliome analyses have high diagnostic yield. Based on our findings we conclude that for new patients with Kabuki-like phenotypes it is possible to choose a specific molecular testing approach that has the highest detection rate for a given MLL2-Kabuki score, thus fostering more precise patient diagnosis and improved management in these genetically- and phenotypically heterogeneous clinical entities.

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".

An ADP-ribosyltransferase 3 (ART3) variant is associated with reduced sperm counts in Czech males: case/control association study replicating results from the Japanese population.

OBJECTIVES: In about 50% of male infertility the underlying pathogenesis remains unknown. A recent Japanese study provided evidence that the rs6836703: G>A single-nucleotide polymorphism (SNP) from the ADP-ribosyltransferase 3 (ART3) gene is significantly associated with non-obstructive azoospermia. However, the functional significance of this association is unknown and replication studies in unrelated populations are thus necessary. DESIGN: In this study, 257 fertile Czech controls of proven paternity and 98 sub-/infertile patients selected according to stringent exclusion / inclusion criteria were genotyped by High Resolution Melting (HRM) of small amplicons. SETTING: This study was performed at University Hospital Motol - Laboratory of reproductive genetics using routinely analyzed cases. RESULTS: Significant differences in allele distribution between fertile and sub-/infertile men were found (OR=1.78, 95% CI: 1.17-2.70; p=0.007). Following sub-stratification of cases according to their sperm counts we found that observed differences in allele distributions were increased in oligozoospermic men with sperm counts of <15 million sperm/mL (OR=1.98, 95% CI: 1.28-3.07; p=0.002). This difference was also reflected in genotype distributions between fertile and sub-/infertile men (p=0.008), and fertile versus oligozoospermic men (p=0.004). CONCLUSIONS: Our study serves as a first replication of the original Japanese report and opens new avenues of research. Compared to the Japanese patient cohort, we provided evidence that the analyzed ART3 variant is associated with quantitative impairment of spermatogenesis.

Diagnostic method validation: High resolution melting (HRM) of small amplicons genotyping for the most common variants in the MTHFR gene.

OBJECTIVES: According to OECD guidelines methods implemented in a diagnostic laboratory should be properly validated prior their implementation. For this purpose we selected genotyping by High Resolution Melting (HRM) of small amplicons using common variants in MTHFR as a model. DESIGN AND METHODS: We selected previously typed samples on which selected analytical validation-related parameters relevant to DNA diagnostics - specificity, sensitivity, precision, robustness and ability to perform reliable calls were evaluated. RESULTS: Correct genotype was assigned in 375/381 (98.4%) for c.677 C>T (rs1801133: C>T; p.A222 V) and in 102/104 (98.1%) for c.1298 A>C (rs1801131: A>C; p.E429A) of all cases. Low analytical failure rate and very high specificity/sensitivity were achieved. Similarly, precision and robustness were consistent. CONCLUSIONS: We have successfully validated HRM of small amplicons using common MTHFR variants as a model. We proved that this technique is highly reliable for routine diagnostics and our diagnostic validation strategy can serve as a model for other applications.

An eleven amino acid residue deletion expands the substrate specificity of acetyl xylan esterase II (AXE II) from Penicillium purpurogenum.

The soft-rot fungus Penicillium purpurogenum secretes to the culture medium a variety of enzymes related to xylan biodegradation, among them three acetyl xylan esterases (AXE I, II and III). AXE II has 207 amino acids; it belongs to family 5 of the carbohydrate esterases and its structure has been determined by X-ray crystallography at 0.9 A resolution (PDB 1G66). The enzyme possesses the alpha/beta hydrolase fold and the catalytic triad typical of serine esterases (Ser90, His187 and Asp175). AXE II can hydrolyze esters of a large variety of alcohols, but it is restricted to short chain fatty acids. An analysis of its three-dimensional structure shows that a loop that covers the active site may be responsible for this strict specificity. Cutinase, an enzyme that hydrolyzes esters of long chain fatty acids and shows a structure similar to AXE II, lacks this loop. In order to generate an AXE II with this broader specificity, the preparation of a mutant lacking residues involving this loop (Gly104 to Ala114) was proposed. A set of molecular simulation experiments based on a comparative model of the mutant enzyme predicted a stable structure. Using site-directed mutagenesis, the loop's residues have been eliminated from the AXE II cDNA. The mutant protein has been expressed in Aspergillus nidulans A722 and Pichia pastoris, and it is active towards a range of fatty acid esters of up to at least 14 carbons. The availability of an esterase with broader specificity may have biotechnological applications for the synthesis of sugar esters.