Abstract
Introduction
ALPK3 null variants (ALPK3nv) in simple heterozygosis represent an established and emerging genetic cause for hypertrophic cardiomyopathy. Some studies have indicated that this ...genetic substrate would be associated with a disease of late-onset disease and incomplete penetrance.
Purpose
To perform an extended genotype-phenotype correlation analysis with respect to simple heterozygous ALPK3nv carriers, focusing on age at diagnosis, maximum wall thickness and survival analysis, performed on the largest available released cohort to date.
Methods
ALPK3 gene was included in an extended NGS panel in 16,780 probands from a reference multicentric genetic laboratory, of whom 6,505 were referred with a HCM phenotype. We collected a composite cohort of ALPK3nv (considering frameshift, nonsense and splice-site predicted frameshift variants), including index and familial cases referred to this laboratory, but also cases reported in the literature. Phenotypic behavior was analyzed, mainly considering age at diagnosis, maximum LV wall thickness, and a composite of events (heart failure and arrhythmia-related endpoints).
Results
A heterozygous ALPK3 null variant was identified in 110 probands out of 6,505 index cases diagnosed with HCM in our center (1.7% of HCM cases). For genotype-phenotype analysis, in total 189 heterozygous carriers were included, considering relatives and cases from the literature; 21 carriers had biallelic involvement (were homozygous or compound heterozygous for two ALPK3Knv).
Among ALPK3nv heterozygous carriers, mean age at diagnosis was 53.4 (±15.2) years; mean left ventricular thickness was 17.7 (±4.9) mm, and morphological profile was mainly apical (42%) and/or concentric (26%). Males tended to be diagnosed at an earlier age than females (median age of diagnosis was 56 vs 71 years, respectively, p <0.01). Five events were observed in this group (four males and one female) all of them related to heart failure, and occur in carriers older than 45 years. The clinical profile was drastically different in cases with biallelic involvement, with a mean age at diagnosis of 22.2±9.3, mean LV wall thickness of 24 mm (±8.6), with LV dysfunction observed in 25%. Nine events were observed in this group, also mostly associated with HF (70%).
Conclusions
ALPK3 null variants in simple heterozygosis can explain 1.7% of HCM. They were associated with late-onset disease, especially in females, predominance of apical and/or concentric forms, and a phenotype that was not particularly severe, both morphologically and in terms of survival.
Abstract
Background/Introduction
Dilated cardiomyopathy (DCM) is one of the most common cardiomyopathies, with a prevalence of 1 in 250. Single nucleotide variants (missense, nonsense, or frameshift) ...in definitive DCM genes are the most frequently identified in positive genetic studies. Despite major advances in genetic diagnostic technologies in recent decades, and more than 100 genes potentially associated with DCM identified to date, the diagnostic yield is still lower than 40%. Copy number variants (CNVs) have been identified as cause of the disease in several studies, especially in genes intolerant to haploinsufficiency. However, the contribution of CNVs to the etiology of DCM has not been evaluated in large cohorts of DCM patients and remains unknown.
Purpose
To determine the percentage of cases in which CNVs have been identified as the cause of disease in a large cohort of DCM patients evaluated genetically.
Methods
Retrospective study in which the presence of CNVs was evaluated in a cohort of patients diagnosed with DCM and studied by next-generation-sequencing (NGS). CNVs were detected using a read depth approach and confirmed by an orthogonal molecular technique (Sanger sequencing, MLPA, or dPCR).
Results
6,512 DCM patients referred for genetic testing were sequenced in our center by NGS, and the median age at the time of genetic study was 51.1 years (range 0-93). A pathogenic (P) or likely-pathogenic (LP) disease-causing variant was identified in 1,495 of the patients (diagnostic yield of 22.9%). In 67 (1.02%) of the cases, a CNV classified as P/LP was identified. DMD was the gene with the highest number of CNVs identified (n=40), followed by TTN (n=7) and LMNA (n=5). Of the 67 cases, 45 (67%) corresponded to deletions, with a lesser extent to duplications (n=9; 13%), complex alterations (n=7; 11%), and deletion/insertion (n=6; 9%). Only two of the patients harboring disease-causing CNVs had other variants potentially associated with DCM: a likely-pathogenic variant in MYH7 and a homozygous pathogenic variant in the ALMS1 gene.
Conclusion
In our cohort of DCM patients, 1.02% harbored CNVs considered P/LP, being DMD the most frequently identified gene. These results show that systematic CNVs analysis in NGS studies improves the yield of genetic testing in DCM and highlights the importance of a complete genetic study that includes structural variant detection in the diagnosis of the disease.
The fluorescence detection of ultra high energy (≳10
18
eV) cosmic rays requires a detailed knowledge of the fluorescence light emission from nitrogen molecules, which are excited by the cosmic ray ...shower particles along their path in the atmosphere. We have made a precise measurement of the fluorescence light spectrum excited by MeV electrons in dry air. We measured the relative intensities of 34 fluorescence bands in the wavelength range from 284 to 429
nm with a high resolution spectrograph. The pressure dependence of the fluorescence spectrum was also measured from a few hPa up to atmospheric pressure. Relative intensities and collisional quenching reference pressures for bands due to transitions from a common upper level were found in agreement with theoretical expectations. The presence of argon in air was found to have a negligible effect on the fluorescence yield. We estimated that the systematic uncertainty on the cosmic ray shower energy due to the pressure dependence of the fluorescence spectrum is reduced to a level of 1% by the AIRFLY results presented in this paper.
Abstract
Background
Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder, with a prevalence between 1/250-500 individuals. The yield of genetic testing for this condition ...stands at 40%. The most prevalent genes are MYBPC3 and MYH7, with small contributions of other genes, such as TNNT2, TNNI3, TPM1, MYL2 or FHOD3. At our center, we have identified a recurrent variant in TNNT2, p.Asn271Ile, associated with HCM. Most probands came from the northwest of Spain (Galicia), which suggested that they may share a common origin.
Purpose
We aimed to determine whether carriers of the p.Asn271Ile variant in TNNT2 share a common haplotype, in order to estimate the age of the mutation and describe the associated phenotype.
Methods
We analyzed probands who had TNNT2 sequenced by NGS at our center. Enrichment of the variant was observed in the HCM cohort. Eight highly polymorphic microsatellite markers flanking the variant in 18 Galician HCM probands were also analyzed, compromising a region of approximately 7,300 kb. Custom PCR assays were designed, and fragment length analysis was performed by capillary electrophoresis.
Results
TNNT2 p.Asn271Ile was identified in 49/28,802 probands (0.17%). The variant was significantly enriched in HCM probands (47/11,870; 0.39%) compared to internal non-HCM controls (2/14,804; 0.02%), with an OR=29.3 (IC95%=7.1-120.7). The analysis revealed that the 18 HCM Galician probands shared a common haplotype of 500 kb, estimating that p.Asn271Ile has arisen approximately 26.5 generations ago (650 years).
Regarding genotype-phenotype correlation, our data (more than 180 carriers from 64 families) shows a relatively benign behavior, with an advance mean age at diagnosis (49.8 (±17.7), non-severe left ventricular hypertrophy 15.3 (±5.3), and a lower rate of major events among carriers of p.Asn271Ile in TNNT2, compared to both other genetic variants in TNNT2 and the main genetic cause of HCM (i.e., null variants in MYBPC3) (p=0.0001 for both comparisons). The prevalence of left ventricular systolic dysfunction and left ventricular tract obstruction was also smaller than that described for other sarcomeric genes (3.54% and 12%, respectively).
Conclusions
A founder effect of the pathogenic variant p.Asn271Ile has been demonstrated, with the variant arising approximately 650 years ago. The clinical behavior in carriers of this cohort showed to be relatively benign compared to the behavior of other genetic variants in this gene and null variants in MYBPC3.
Abstract
Introduction
MYH7 encodes to β-cardiac myosin heavy chain, a large and pleomorphic protein, conformed by different and well-functionally characterized domains. This gene has an enormous ...contribution to hypertrophic cardiomyopathy (HCM) but also to other cardiomyopathies such as dilated, non-compaction and restrictive cardiomyopathy (and overlapping phenotypes). There are specific and adapted recommendations for classifying variants in this gene regarding its association with HCM; one of the aspects was the location of variants in the head domain, encompassing 181–937 residues as moderate ACMG criteria (Kelli et al., 2018).
Objectives
The purpose of this analysis was to determine if more specific regions of MYH7are enriched in HCM cases. This analysis could help to better characterise those regions where identifying a novel missense variant could reinforce their pathogenicity.
Methods
We included in the analysis 26,929 consecutives unrelated probands in which MYH7 was sequenced by NGS, referred from different countries and centres. We performed an enrichment analysis comparing the prevalence of rare missense variants (pre-established MAF cut-off 0.004%) in cases with the diagnosis of HCM versus their frequency in non-cardiomyopathy cases (internal controls, OR_int, comprising aortic diseases, channelopathies and dyslipidaemias) and in gnomAD population (external controls, OR_ext). Comparison between groups was performed using the Student's t-test; p value <0.05 was considered to indicate statistical significance. The rationale of this grouping was to avoid skewed analysis, given the high degree of overlapping cardiomyopathies observed in this gene.
Results
HCM was the diagnosis in 10,064 cases. 8,975 probands with a non-cardiomyopathy phenotype were used as internal controls; 7,890 cases with other MYH7-related phenotypes (restrictive, dilated, non-compaction and skeletal myopathies) were not included in the analysis. Rare missense variants in MYH7 were enriched in HCM cases (OR_int = 11.15; OR_ext =12.44) and in the pre-established hot spot head domain (OR_int = 16.43; OR_ext = 24.53). However, the regions with the highest odds ratios in both external and internal comparison were the central converter domain (residues 711–755, OR_int = 44.39; OR_ext = 50.27) and actin-binding region (residues 647–664, OR_int =18.77; OR_ext = 69.32). Conversely, the tail region (both S2 subfragment and light meromyosin region, LMM), had the lowest OR in this analysis.
Conclusions
Previously reported hotspot (head) is significantly enriched in HCM cases, but some specific regions within this domain, such as central converter and acting-binding region, could have a relatively higher contribution. These results reinforce the current approach, but on the other hand, show an asymmetric contribution within the head domain. This fact may have an impact on the reassessment of the variant classification in this gene towards a more precise approach.
Funding Acknowledgement
Type of funding sources: Private company. Main funding source(s): Health in Code
Abstract Background Copy number variations (CNVs) have increasingly been recognized in the cardiovascular setting. Their role in hypertrophic cardiomyopathy or sudden cardiac death has been ...described, but their involvement in Long QT syndrome (LQTS) has been scarcely studied. Most of these descriptions are confined to case reports or small series. Purpose The aim of this study is to investigate the proportion of clinically relevant CNVs among a large cohort of index cases referred for genetic testing due to LQTS. Methods Retrospective study in which the presence of likely pathogenic (LP) or pathogenic (P) CNVs was evaluated in a cohort of index cases referred for genetic study with LQTS phenotype. Index cases were genotyped with customized libraries through next generation sequencing (NGS). CNVs analysis was conducted by a proprietary method by means of comparative analysis of normalized read depth data. Clinically relevant (LP or P) CNVs were then confirmed by other techniques. Results 1183 index cases were referred for genetic testing to our laboratory with long QT syndrome phenotype. Some of these index cases were referred for suspicion of Jervell-Lange-Nielsen syndrome (JNLS) or for LQTS associated with congenital deafness (n=10) or Anderson-Tawil syndrome (n=1). Around 27% (n=322) of the index cases had a positive result. CNVs analysis was run on 1071 (91%) of the index cases. Most of the CNVs were considered negative (n=994, 93%), 59 could not be analysed (6%) and in 11 patients (1%), a LP/P CNV was identified. All CNVs but three (72.72%), were confirmed by orthogonal molecular techniques (Sanger sequencing or multiplex ligation-dependent probe amplification (MLPA)). In all, but two, the genetic study was considered positive due to the CNV findings. One of these patients carried a pathogenic lost-of-function (LOF) CNV in the LDLR gene (incidental finding) but the report was negative because he was referred for suspicion of JLNS. In another case a CNV in the DSP gene was identified in mosaicism (30-40%). However, in this case, the positive report was attributed to the presence of an additional pathogenic variant in CACNA1C. Regarding the other index cases with LP/P CNVs: 4 carried a deletion in the KCNH2 gene, 3 carried a deletion in KCNQ1 gene (one of them in homozygosis and was studied for suspicion of JLNS) and 1 carried a duplication in the KCQN1 (LOF variant). In addition, one patient carried a deletion of exon 3 in the RYR2 gene. Even if the patient was referred for suspicion of LQTS, the report was considered positive. These clinically relevant CNVs represented around 3% of the positive genetic results. Conclusions In our large cohort of patients referred for genetic testing for LQTS, around 1% of clinically relevant CNVs were identified. As such large genomic rearrangements underlie a non-neglectable portion of cases, we consider that their analysis should be performed as part of the routine genetic testing.Deletion exons 6-14 in KCNH2 gene (CNV)MLPA with deletion exons 6-14 in KCNH2
Abstract Background Diagnostic yield in long QT syndrome (LQTS) ranges between 30-70%, depending on the studied population. LQTS can lead to an unexpected sudden cardiac death. For this reason and ...due to its wider current availability, genetic testing in patients with suspicion of LQTS has spread. Purpose The aim of this study is to investigate the genetic testing yield among a large cohort of heterogeneous index cases with LQTS phenotype. Methods Diagnostic yield was restrospectively evaluated in a cohort of index cases referred to our laboratory for genetic study with LQTS phenotype. Genetic study was performed with customized libraries through next generation sequencing (NGS), which included copy number variations (CNVs) analysis. Patients with LQTS phenotype studied both with "long QT panels" and with broader cardiovascular panels were included, but those with additional cardiac phenotype (cardiomyopathies, heart defects or catecholaminergic polymorphic ventricular tachycardia) were excluded. Positive genetic reports were considered when a likely pathogenic (LP) or pathogenic (P) variant could explain patient´s phenotype. Inconclusive reports were described when a variant considered risk factor (RF) or a variant of uncertain significance variant (VUS) or a hot-VUS was identified in a clinically relevant gene. Results 1183 index cases were genotyped for LQTS phenotype. Only 164 index cases (13,8%) were studied with broader panels which included between 77 and 368 genes. The rest (n=1019) were studied with LQTS panels (either with small panels which included KCNQ1, KCNH2, KCNE1, KCNE2, SCN5A, KCNJ2, CACNA1C and with up to 36 genes-QT panels). Around 27% (n=321) of the index cases had a positive result. They carried LP/P variants in the following genes: KCNQ1 (n=166, 52%), KCNH2 (n=112), SCN5A (n=25), RYR2 (n=5), CACNA1C (n=3), HCN4 (n=4), KCNJ2 (n=2), KCNE2 (n=2) and CALM2 (n=1). 53,3% of the reports were negative. However, there were some incidental findings: 2 index cases carried P variants in LDLR, 2 carried P variants in PKP2, 2 carried a P variant in MYBPC3 and 1 index case each carried a P variant in DES, TTN and DSG2. From the 231 inconclusive reports, 37 index cases carried the RF variant in KCNE1 (p.Asp85Asn), 2 carried the RF variant in SCN5A (p.Arg1193Gln) and 50 patients carried hot-VUS in KCNQ1, KCNH2, CACNA1C, SCN5a or RYR2 gene. Conclusions In our large cohort of index cases referred for genetic testing for LQTS, diagnostic yield was around 27% when considering only LP/P variants in LQTS or arrhythmic phenotype related genes. Even by including the inconclusive but potentially relevant genetic results (RF and hot VUS), the diagnostic yield in our cohort reaches 35%. This is lower than expected for LQTS cohort. It should be taken into consideration that our cohort consists of heterogeneous patients referred for LQTS genetic testing, which would probably reflects better the real world patients seen in every day practice.
We develop an algorithm that has the potential to relate the depth development of ultra high energy extensive air showers and the time delay for individual muons. The time distributions sampled at ...different positions at ground level by a large air shower array are converted into distributions of production distances using an approximate relation between production distance, transverse distance and time delay. The method is naturally restricted to inclined showers where muons dominate the signal at ground level but could be extended to vertical showers provided that the detectors used can separate the muon signal from electrons and photons. We explore the accuracy and practical uncertainties involved in the proposed method. For practical purposes only the muons that fall outside the central region of the shower can be used, and we establish cuts in transverse distance. The method is tested using simulated showers by comparing the production distance distributions obtained using the method with the actual distances in the simulated showers. It could be applied in the search for neutrinos to increase the acceptance to highly penetrating particles, as well as for unraveling the relative compositions of protons and heavy nuclei. We also illustrate that the obtained depth distributions have minimum width when both the arrival direction and the core position are well reconstructed.
An analytical description of the time structure of the pulses induced by muons in air showers at ground level is deduced assuming the production distance distribution for the muons can be obtained ...elsewhere. The results of this description are compared against those obtained from simulated showers using AIRES. Major contributions to muon time delays are identified and a relation between the time structure and the depth distribution is unveiled.
The fluorescence detection of ultra high energy cosmic rays requires a detailed knowledge of the fluorescence light emission from nitrogen molecules over a wide range of atmospheric parameters, ...corresponding to altitudes typical of the cosmic ray shower development in the atmosphere. We have studied the temperature and humidity dependence of the fluorescence light spectrum excited by MeV electrons in air. Results for the 313.6, 337.1, 353.7 and 391.4
nm bands are reported in this paper. We found that the temperature and humidity dependence of the quenching process changes the fluorescence yield by a sizeable amount (up to 20% for the temperature dependence in the 391.4
nm band) and its effect must be included for a precise estimation of the energy of ultra high energy cosmic rays.
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