Correction to: Usefulness of Genetic Testing in Hypertrophic Cardiomyopathy: an Analysis Using Real-World Data

post-print123 K

Usefulness of genetic testing in hypertrophic cardiomyopathy. An analysis using real-world data.

Aims: This study sought to determine the usefulness of genetic testing to predict evolution in hypertrophic cardiomyopathy (HCM) and to assess the role of genetic testing in clinical practice. Methods and Results: Genetic results of 100 HCM patients tested for mutations in ≥10 HCM-causing genes were evaluated. Patients were classified as with poor (Group A) or favourable(Group B) clinical course. Forty-five pathogenic mutations (PM) were identified in 28 patients (56%) from Group A and in 23 (46%) from Group B (p=0.317). Only 40 patients (40%) exhibited PM that had been previously reported and only 15 (15%) had PM reported in ≥10 individuals. PM associated with poor prognosis were identified in just 5 patients from Group A (10%). Conclusion: Genetic findings are not useful to predict prognosis in most HCM patients. By contrast, real-world data reinforce the usefulness of genetic testing to provide genetic counselling and to enable cascade genetic screening.pre-print298 K

Community-acquired methicillin-resistant Staphylococcus aureus: what do we need to know?

AbstractCommunity-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has become a matter of concern worldwide, in particular in the USA. For the analysis of emergence and spread, clear definitions based on epidemiological origin are needed for discrimination between CA-MRSA, healthcare-associated community MRSA, and healthcare-associated MRSA (HA-MRSA). Although its role in pathogenesis is currently under debate, the capability for Panton–Valentine leukocidin formation is associated with the majority of CA-MRSA isolates from North America and from Europe. Most CA-MRSA isolates are attributed to clonal lineages different from HA-MRSA; there are, however, clonal lineages from which both HA-MRSA and CA-MRSA have been reported (e.g. ST1, ST5, ST8, and ST22); CA-MRSA ST8 (USA300), which is most frequent in the USA, has meanwhile been reported from Europe. CA-MRSA ST80 is widely disseminated in Europe; because of its pronounced oxacillin heteroresistance phenotype, cefoxitin-based assays are advisable for reliable detection. So far, CA-MRSA infections seem to be much less frequent in Europe than in the USA, where patients with particular predispositions and low social status are at especial risk

Large Genomic Imbalances in Brugada Syndrome

Purpose Brugada syndrome (BrS) is a form of cardiac arrhythmia which may lead to sudden cardiac death. The recommended genetic testing (direct sequencing of SCN5A) uncovers disease-causing SNVs and/or indels in ~20% of cases. Limited information exists about the frequency of copy number variants (CNVs) in SCN5A in BrS patients, and the role of CNVs in BrS-minor genes is a completely unexplored field. Methods 220 BrS patients with negative genetic results were studied to detect CNVs in SCN5A. 63 cases were also screened for CNVs in BrS-minor genes. Studies were performed by Multiplex ligation-dependent probe amplification or Next-Generation Sequencing (NGS). Results The detection rate for CNVs in SCN5A was 0.45% (1/220). The detected imbalance consisted of a duplication from exon 15 to exon 28, and could potentially explain the BrS phenotype. No CNVs were found in BrS-minor genes. Conclusion CNVs in current BrS-related genes are uncommon among BrS patients. However, as these rearrangements may underlie a portion of cases and they undergo unnoticed by traditional sequencing, an appealing alternative to conventional studies in these patients could be targeted NGS, including in a single experiment the study of SNVs, indels and CNVs in all the known BrS-related genes

Natural and Undetermined Sudden Death: Value of Post-Mortem Genetic Investigation

Background: Sudden unexplained death may be the first manifestation of an unknown inherited cardiac disease. Current genetic technologies may enable the unraveling of an etiology and the identification of relatives at risk. The aim of our study was to define the etiology of natural deaths, younger than 50 years of age, and to investigate whether genetic defects associated with cardiac diseases could provide a potential etiology for the unexplained cases. Methods and Findings: Our cohort included a total of 789 consecutive cases (77.19% males) <50 years old (average 38.6±12.2 years old) who died suddenly from non-violent causes. A comprehensive autopsy was performed according to current forensic guidelines. During autopsy a cause of death was identified in most cases (81.1%), mainly due to cardiac alterations (56.87%). In unexplained cases, genetic analysis of the main genes associated with sudden cardiac death was performed using Next Generation Sequencing technology. Genetic analysis was performed in suspected inherited diseases (cardiomyopathy) and in unexplained death, with identification of potentially pathogenic variants in nearly 50% and 40% of samples, respectively. Conclusions: Cardiac disease is the most important cause of sudden death, especially after the age of 40. Close to 10% of cases may remain unexplained after a complete autopsy investigation. Molecular autopsy may provide an explanation for a significant part of these unexplained cases. Identification of genetic variations enables genetic counseling and undertaking of preventive measures in relatives at risk

Personalized Interpretation and Clinical Translation of Genetic Variants Associated With Cardiomyopathies

Cardiomyopathies are a heterogeneous group of inherited cardiac diseases characterized by progressive myocardium abnormalities associated with mechanical and/or electrical dysfunction. Massive genetic sequencing technologies allow a comprehensive genetic analysis to unravel the cause of disease. However, most identified genetic variants remain of unknown clinical significance due to incomplete penetrance and variable expressivity. Therefore, genetic interpretation of variants and translation into clinical practice remain a current challenge. We performed retrospective comprehensive clinical assessment and genetic analysis in six families, four diagnosed with arrhythmogenic cardiomyopathy, and two diagnosed with hypertrophic cardiomyopathy (HCM). Genetic testing identified three rare variants (two non-sense and one small indel inducing a frameshift), each present in two families. Although each variant is currently classified as pathogenic and the cause of the diagnosed cardiomyopathy, the onset and/or clinical course differed in each patient. New genetic technology allows comprehensive yet cost-effective genetic analysis, although genetic interpretation, and clinical translation of identified variants should be carefully done in each family in a personalized manner

Estratègies de diagnòstic genètic en fetus amb malformacions congènites. Correlació genotip-fenotip

[cat] Les alteracions cromosòmiques constitucionals representen una de les principals causes d’anomalies congènites a la població, i el seu diagnòstic durant l’etapa prenatal és el principal objectiu de la majoria de procediments invasius que es realitzen durant l’embaràs. Actualment, el cariotip convencional és el gold standard del diagnòstic citogenètic prenatal, però els avanços tecnològics dels últims anys han portat al desenvolupament de tècniques de citogenètica molecular que ofereixen característiques molt atractives, la principal de les quals és l’elevada resolució. Donat que el paper que han de tenir aquestes tècniques durant l’etapa prenatal encara no està ben definit, els objectius de la present tesi han estat: a) determinar el potencial diagnòstic i la utilitat del cariotip i les tècniques de citogenètica molecular Fluorescent In Situ Hybridization (FISH), Multiplex Ligation-dependent Probe Amplification (MLPA) i Chromosomal Microarray-based Analysis (CMA) en diagnòstic prenatal, especialment per a l’estudi de gestacions amb troballes ecogràfiques; i b) valorar la necessitat de modificar els procediments actuals de diagnòstic citogenètic prenatal. En primer lloc, s’han revisat els resultats dels 29.883 cariotips en líquid amniòtic realitzats entre 1998 i 2009 a l’Hospital Clínic de Barcelona. Els resultats obtinguts corroboren que el cariotip és una eina efectiva i robusta per a l’obtenció del cariotip fetal, revelant alteracions cromosòmiques en el 2,9% de les gestacions que es sotmeten a un procediment invasiu. La translucidesa nucal incrementada i les anomalies ecogràfiques destaquen com a indicadors excel•lents d’anomalia cromosòmica. En segon lloc, s’ha avaluat la utilitat de l’MLPA subtelomèrica per a l’estudi de gestacions amb troballes ecogràfiques i cariotip normal. Dels 229 fetus analitzats, 3 presentaven desequilibris subtelomèrics críptics (1,3%). Donat que la freqüència d’aquests desequilibris és baixa, i a més la correlació genotip-fenotip observada és pobra, l’MLPA subtelomèrica no sembla ser una eina crucial per a l’estudi d’aquestes gestacions. D’altra banda, s’ha estudiat la idoneïtat de reemplaçar els estudis de FISH de la regió 22q11.2 per kits d’MLPA dissenyats per a l’estudi d’aquesta regió i altres regions genòmiques associades a cardiopaties. S’han analitzat mitjançant aquests kits 55 gestacions amb troballes cardíaques, cariotip normal i estudi de FISH negatiu per a la microdeleció típica de 22q11.2, i no s’ha detectat cap anomalia. Aquest fet, juntament amb que l’MLPA presenta una taxa de repetició i de no obtenció de resultats superiors a la FISH, i que generalment requereix cultiu cel•lular, indica que la FISH hauria de seguir sent la tècnica d’elecció per a l’estudi de fetus amb troballes cardíaques (enfront l’MLPA). També s’ha volgut determinar la freqüència dels diferents tipus d’alteracions cromosòmiques, tant microscòpiques com críptiques, en fetus amb troballes ecogràfiques cardíaques. L’anàlisi retrospectiu dels resultats obtinguts en el període 2009-2011 a l’Hospital Clínic de Barcelona en aquest grup de gestacions (N=276) ha revelat una freqüència d’anomalies cromosòmiques microscòpiques i casos de Síndrome de la deleció cromosòmica 22q11.2 del 15,9% i 6,4%, respectivament; uns resultats que corroboren la forta associació existent entre troballes ecogràfiques cardíaques i anomalies cromosòmiques. A més, aquesta associació varia significativament en funció del tipus de troballa ecogràfica cardíaca i de la presència d’anomalies extracardíaques. D’altra banda, s’han analitzat mitjançant array CGH 51 fetus amb troballes ecogràfiques cardíaques, cariotip normal, i sense estudi o resultat de FISH negatiu per a la Sd. de la deleció cromosòmica 22q11.2. En aquest grup de gestacions, la taxa de detecció de variants en número de còpia patogèniques ha estat del 2%, i no s’han detectat variants de significat clínic incert. Si en la nostra sèrie de 276 gestacions amb troballes ecogràfiques cardíaques s’hagués utilitzat l’estratègia QF-PCR + CMA enlloc de cariotip, totes les anomalies cromosòmiques amb repercussió clínica clara diagnosticades per citogenètica convencional s’haguessin identificat, s’haguessin detectat els casos de delecions de 22q11.2, i s’hagués incrementat un 2% el nombre de casos diagnosticats. Per tant, aquests resultats mostren el potencial de les plataformes de microarray per al diagnòstic prenatal de fetus amb troballes ecogràfiques cardíaques. Finalment, s’han utilitzat les tècniques de citogenètica molecular FISH, MLPA i/o CMA per a la caracterització de 6 alteracions cromosòmiques visibles al cariotip però de difícil caracterització per citogenètica convencional. En tots els casos, la utilització d’aquestes tècniques ha contribuït a la correcte descripció de l’alteració identificada, posant de manifest la seva utilitat quan s’utilitzen de forma complementària al cariotip. L’elaboració d’aquesta tesi i la literatura recentment publicada evidencien la necessitat d’un nou plantejament dels protocols clínics de diagnòstic citogenètic prenatal, especialment en relació a la utilització de plataformes de microarray en diagnòstic prenatal[eng] Strategies for genetic diagnosis of fetuses with congenital malformations. Genotype-phenotype correlations. Constitutional chromosomal abnormalities represent one of the main causes of congenital abnormalities in the population, and their diagnosis during pregnancy is the main objective of most invasive procedures. Nowadays, conventional karyotyping is the gold standard for prenatal cytogenetic diagnosis, but recent technological advances have led to the development of molecular cytogenetic techniques which offer much higher resolution. Since there is still not a consensus on the role of these techniques in prenatal diagnosis, the goals of the present PhD work have been: a) to determine the diagnostic potential and usefulness of karyotype and molecular cytogenetic techniques Fluorescent In Situ Hybridization (FISH), Multiplex Ligation-dependent Probe Amplification (MLPA) and Chromosomal Microarray-based Analysis (CMA) in prenatal diagnosis, especially in pregnancies with ultrasound findings; and b) to establish the need for modifications in current prenatal cytogenetic diagnostic protocols. The results obtained: - Corroborate the effectiveness and robustness of conventional karyotyping. The observed detection rate of chromosomal abnormalities in 29,883 consecutive amniotic fluid samples has been 2.9%, with increased nuchal translucency and ultrasound abnormalities being excellent indicators for chromosomal abnormalities. - Suggest that subtelomeric screening in pregnancies with ultrasound findings and normal karyotype is not a crucial tool, as detection rate of subtelomeric imbalances is low (1.3%; 3/229), and genotype-phenotype correlations are poor. - Reveal that FISH of 22q11.2 region is preferred to MLPA kits specific for this chromosome region and other genomic regions previously associated with congenital heart defects. - Reveal a strong association between cardiac ultrasound findings and chromosomal abnormalities, both microscopic and cryptic. In pregnancies with cardiac findings, the observed frequency of microscopic chromosomal abnormalities and chromosome 22q11.2 deletion syndrome has been 15.9% (44/276) and 6.4% (5/78), respectively. Moreover, CMA has been performed in 51 fetuses with cardiac ultrasound findings, normal karyotype and negative or no FISH study for chromosome 22q11.2 deletion syndrome, and the detection rate of pathogenic copy number variants has been 2%, without detection of variants of unknown clinical significance. If in the initial series of 276 pregnancies the strategy QF-PCR + CMA would have been applied; all the chromosomal abnormalities with clear clinical repercussion diagnosed by conventional cytogenetics would have been identified, together with the deletions of 22q11.2 region and an extra 2% of chromosomal abnormalities. These results show the potential of CMA for the prenatal diagnosis of these pregnancies. - Show the usefulness of the molecular cytogenetic techniques FISH, MLPA and CMA when used as a complement to conventional karyotyping in cases with chromosomal abnormalities not accurately characterizable by conventional cytogenetics. Accordingly, the results obtained suggest the need for updating current prenatal cytogenetic diagnostic protocols

Overview of chromosome abnormalities in first trimester miscarriages: a series of 1,011 consecutive chorionic villi sample karyotypes

In order to contribute to the knowledge of type and frequency of chromosome abnormalities in early pregnancy losses, we analyzed the cytogenetic results from a large series of first trimester miscarriages, using a diagnostic approach with a high success rate and no maternal contamination. A total of 1,119 consecutive chorionic villi samples were obtained before evacuation, and karyotypes were prepared after short-term culture (STC). In 603 samples, a long-term culture (LTC) was also performed. The overall and individual frequencies of the different types of chromosome abnormalities were established, including placental mosaicisms, and their relationship with maternal age and gestational weeks was assessed. An abnormal karyotype was detected in 70.3% of the samples. Single autosomal trisomy was the most frequent abnormality (64.6% of the abnormal cases), followed by triploidy (13.1%) and monosomy X (10.4%). Chromosome rearrangements were found in 5.2%, combined abnormalities in 8.9%, and placental mosaicism in 3.5% of the cases with STC and LTC performed. Individual trisomies behaved differently with respect to maternal age and intrauterine survival. Due to the combination of STC and LTC, our study offers reliable information on the incidence and type of chromosome abnormalities and placental mosaicism in miscarriages and contributes to define the cytogenetic implication in their etiology.This work was partially supported by grants PI11/01841 (IP: A. Sánchez) and PI14/00588 (IP: A. Borrell) from Instituto de Salud Carlos III (Spain) and jointly financed by Fondo Europeo de Desarrollo Regional, Union Europea, Una manera de hacer Europa (FEDER).Peer reviewe

Rare Titin (TTN) Variants in Diseases Associated with Sudden Cardiac Death

A leading cause of death in western countries is sudden cardiac death, and can be associated with genetic disease. Next-generation sequencing has allowed thorough analysis of genes associated with this entity, including, most recently, titin. We aimed to identify potentially pathogenic genetic variants in titin. A total of 1126 samples were analyzed using a custom sequencing panel including major genes related to sudden cardiac death. Our cohort was divided into three groups: 432 cases from patients with cardiomyopathies, 130 cases from patients with channelopathies, and 564 post-mortem samples from individuals showing anatomical healthy hearts and non-conclusive causes of death after comprehensive autopsy. None of the patients included had definite pathogenic variants in the genes analyzed by our custom cardio-panel. Retrospective analysis comparing the in-house database and available public databases also was performed. We identified 554 rare variants in titin, 282 of which were novel. Seven were previously reported as pathogenic. Of these 554 variants, 493 were missense variants, 233 of which were novel. Of all variants identified, 399 were unique and 155 were identified at least twice. No definite pathogenic variants were identified in any of genes analyzed. We identified rare, mostly novel, titin variants that seem to play a potentially pathogenic role in sudden cardiac death. Additional studies should be performed to clarify the role of these variants in sudden cardiac death

Identification of Genetic Alterations, as Causative Genetic Defects in Long QT Syndrome, Using Next Generation Sequencing Technology.

BACKGROUND:Long QT Syndrome is an inherited channelopathy leading to sudden cardiac death due to ventricular arrhythmias. Despite that several genes have been associated with the disease, nearly 20% of cases remain without an identified genetic cause. Other genetic alterations such as copy number variations have been recently related to Long QT Syndrome. Our aim was to take advantage of current genetic technologies in a family affected by Long QT Syndrome in order to identify the cause of the disease. METHODS:Complete clinical evaluation was performed in all family members. In the index case, a Next Generation Sequencing custom-built panel, including 55 sudden cardiac death-related genes, was used both for detection of sequence and copy number variants. Next Generation Sequencing variants were confirmed by Sanger method. Copy number variations variants were confirmed by Multiplex Ligation dependent Probe Amplification method and at the mRNA level. Confirmed variants and copy number variations identified in the index case were also analyzed in relatives. RESULTS:In the index case, Next Generation Sequencing revealed a novel variant in TTN and a large deletion in KCNQ1, involving exons 7 and 8. Both variants were confirmed by alternative techniques. The mother and the brother of the index case were also affected by Long QT Syndrome, and family cosegregation was observed for the KCNQ1 deletion, but not for the TTN variant. CONCLUSIONS:Next Generation Sequencing technology allows a comprehensive genetic analysis of arrhythmogenic diseases. We report a copy number variation identified using Next Generation Sequencing analysis in Long QT Syndrome. Clinical and familiar correlation is crucial to elucidate the role of genetic variants identified to distinguish the pathogenic ones from genetic noise