Genetic investigations of 100 inherited cardiac disease-related genes in deceased individuals with schizophrenia.

Cardiac diseases and sudden cardiac death (SCD) are more prevalent in individuals diagnosed with schizophrenia compared to the general population, with especially coronary artery disease (CAD) as the major cardiovascular cause of death. Antipsychotic medications, genetics, and lifestyle factors may contribute to the increased SCD in individuals with schizophrenia. The role of antipsychotic medications and lifestyle factors have been widely investigated, while the genetic predisposition to inherited cardiac diseases in schizophrenia is poorly understood. In this study, we examined 100 genes associated with inherited cardiomyopathies and cardiac channelopathies in 97 deceased individuals diagnosed with schizophrenia for the prevalence of genetic variants associated with SCD. The deceased individuals had various causes of death and were included in the SURVIVE project, a prospective, autopsy-based study of mentally ill individuals in Denmark. This is the first study of multiple inherited cardiac disease-related genes in deceased individuals with diagnosed schizophrenia to shed light on the genetic predisposition to SCD in individuals with schizophrenia. We found no evidence for an overrepresentation of rare variants with high penetrance in inherited cardiac diseases, following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG) consensus guidelines. However, we found that the deceased individuals had a statistically significantly increased polygenic burden caused by variants in the investigated heart genes compared to the general population. This indicates that common variants with smaller effects in heart genes may play a role in schizophrenia.

Targeted molecular genetic testing in young sudden cardiac death victims from Western Denmark.

Sudden unexpected death in the young continues to be an important unsolved challenge. A significant proportion of the deaths are suspected to be caused by inherited cardiac diseases and are referred to as sudden cardiac deaths (SCD). We performed targeted molecular testing of 70 deceased individuals under 40 years of age that after forensic autopsy were suspected to have died of SCD. The individuals were previously genetically investigated using smaller numbers of genes associated with specific cardiac diseases. In our previous studies, seven (10%) individuals had pathogenic or likely pathogenic variants according to the 2015 ACMG guidelines. In order to investigate the value of expanding the panel to 100 genes associated with cardiac diseases, we histopathologically re-examined the 70 suspected SCD cases and grouped them according to phenotypes into suspected cardiomyopathy (the cardiomyopathy group), left ventricular hypertrophy (the hypertrophy group) and structural normal hearts (the SUD group). DNA was captured with the Haloplex target enrichment system and sequenced using an Illumina MiSeq. We found that 11 (16%) individuals harboured pathogenic or likely pathogenic variants. In the cardiomyopathy, hypertrophy and SUD groups, 22%, 6% and 17% of the individuals, respectively, harboured pathogenic or likely pathogenic variants. Our findings show that testing of a broad panel of genes associated with cardiac diseases identify potential pathogenic variants of cardiac diseases in a significant proportion of SCD cases, and this may have important implications in family screening to prevent future deaths.

Targeted exon sequencing in deceased schizophrenia patients in Denmark.

Schizophrenia patients have higher mortality rates and lower life expectancy than the general population. However, forensic investigations of their deaths often fail to determine the cause of death, hindering prevention. As schizophrenia is a highly heritable condition and given recent advances in our understanding of the genetics of schizophrenia, it is now possible to investigate how genetic factors may contribute to mortality. We made use of findings from genome-wide association studies (GWAS) to design a targeted panel (PsychPlex) for sequencing of exons of 451 genes near index single nucleotide polymorphisms (SNPs) identified with GWAS. We sequenced the DNA of 95 deceased schizophrenia patients included in SURVIVE, a prospective, autopsy-based study of mentally ill persons in Denmark. We compared the allele frequencies of 1039 SNPs in these cases with the frequencies of 2000 Danes without psychiatric diseases and calculated their deleteriousness (CADD) scores. For 81 SNPs highly associated with schizophrenia and CADD scores above 15, expression profiles in the Genotype-Tissue Expression (GTEx) Project indicated that these variants were in exons, whose expressions are increased in several types of brain tissues, particularly in the cerebellum. Molecular pathway analysis indicated the involvement of 163 different pathways. As for rare SNP variants, most variants were scored as either benign or likely benign with an average of 17 variants of unknown significance per individual and no pathogenic variant. Our results highlight the potential of DNA sequencing of an exon panel to discover genetic factors that may be involved in the development of schizophrenia.

Non-invasive prenatal paternity testing using a standard forensic genetic massively parallel sequencing assay for amplification of human identification SNPs.

Prenatal paternity testing often relies on invasive procedures that cause risk to both the mother and the foetus. Non-invasive, prenatal paternity testing by investigating paternally inherited single nucleotide polymorphisms (SNPs) in cell-free foetal DNA (cffDNA) in maternal plasma was performed at consecutive time points during early gestation. Plasma from 15 pregnant women was investigated at consecutive time points from gestational weeks (GWs) 4-20. The Precision ID Identity Panel and an Ion S5 Sequencer was used to analyse the cffDNA. Paternally inherited foetal SNP alleles were detected from GW7. The median foetal fractions were 0%, 3.9%, 5.1%, 5.2%, and 4.7% at GWs 4, 7, 12, 16, and 20, respectively. The corresponding median numbers of detected paternally inherited foetal autosomal SNP alleles were 0, 3, 9, 10, and 12, respectively. The typical (i.e. geometric mean) paternity indices at GW12 and GW20 were 24 (range 0.0035-8389) and 199 (range 5.1-30,137), respectively. The method is very promising. However, the method can be improved by shortening the lengths of the PCR amplicons and increasing the number of SNPs. To our knowledge, this is the first study to successfully identify paternally inherited foetal SNP alleles at consecutive time points in early gestation independently of the foetal gender.

Sequence variants in muscle tissue-related genes may determine the severity of muscle contractures in cerebral palsy.

Muscle contractures are a common complication to cerebral palsy (CP). The purpose of this study was to evaluate whether individuals with CP carry specific gene variants of important structural genes that might explain the severity of muscle contractures. Next-generation-sequencing (NGS) of 96 candidate genes associated with muscle structure and metabolism were analyzed in 43 individuals with CP (Gross Motor Function classification system [GMFCS] I, n=10; GMFCS II, n=14; GMFCS III, n=19) and four control participants. In silico analysis of the identified variants was performed. The variants were classified into four categories ranging from likely benign (VUS0) to highly likely functional effect (VUS3). All individuals with CP were classified and grouped according to their GMFCS level: Statistical comparisons were made between GMFCS groups. Kruskal-Wallis tests showed significantly more VUS2 variants in the genes COL4 (GMFCS I-III; 1, 1, 5, respectively [p < .04]), COL5 (GMFCS I-III; 1, 1, 5 [p < .04]), COL6 (GMFCS I-III; 0, 4, 7 [p < .003]), and COL9 (GMFCS I-III; 1, 1, 5 [p < .04]), in individuals with CP within GMFCS Level III when compared to the other GMFCS levels. Furthermore, significantly more VUS3 variants in COL6 (GMFCS I-III; 0, 5, 2 [p < .01]) and COL7 (GMFCS I-III; 0, 3, 0 [p < .04]) were identified in the GMFCS II level when compared to the other GMFCS levels. The present results highlight several candidate gene variants in different collagen types with likely functional effects in individuals with CP.

A comparative study of single nucleotide variant detection performance using three massively parallel sequencing methods.

Massively parallel sequencing (MPS) has revolutionised clinical genetics and research within human genetics by enabling the detection of variants in multiple genes in several samples at the same time. Today, multiple approaches for MPS of DNA are available, including targeted gene sequencing (TGS) panels, whole exome sequencing (WES), and whole genome sequencing (WGS). As MPS is becoming an integrated part of the work in genetic laboratories, it is important to investigate the variant detection performance of the various MPS methods. We compared the results of single nucleotide variant (SNV) detection of three MPS methods: WGS, WES, and HaloPlex target enrichment sequencing (HES) using matched DNA of 10 individuals. The detection performance was investigated in 100 genes associated with cardiomyopathies and channelopathies. The results showed that WGS overall performed better than those of WES and HES. WGS had a more uniform and widespread coverage of the investigated regions compared to WES and HES, which both had a right-skewed coverage distribution and difficulties in covering regions and genes with high GC-content. WGS and WES showed roughly the same high sensitivities for detection of SNVs, whereas HES showed a lower sensitivity due to a higher number of false negative results.

Effect of women's age on embryo morphology, cleavage rate and competence-A multicenter cohort study.

This multicenter cohort study on embryo assessment and outcome data from 11,744 IVF/ICSI cycles with 104,830 oocytes and 42,074 embryos, presents the effect of women's age on oocyte, zygote, embryo morphology and cleavage parameters, as well as cycle outcome measures corrected for confounding factors as center, partner's age and referral diagnosis. Cycle outcome data confirmed the well-known effect of women's age. Oocyte nuclear maturation and proportion of 2 pro-nuclear (2PN) zygotes were not affected by age, while a significant increase in 3PN zygotes was observed in both IVF and ICSI (p<0.0001) with increasing age. Maternal age had no effect on cleavage parameters or on the morphology of the embryo day 2 post insemination. Interestingly, initial hCG value after single embryo transfer followed by ongoing pregnancy was increased with age in both IVF (p = 0.007) and ICSI (p = 0.001) cycles. For the first time, we show that a woman's age does impose a significant footprint on early embryo morphological development (3PN). In addition, the developmentally competent embryos were associated with increased initial hCG values as the age of the women increased. Further studies are needed to elucidate, if this increase in initial hCG value with advancing maternal age is connected to the embryo or the uterus.

Genetic investigation of 100 heart genes in sudden unexplained death victims in a forensic setting.

In forensic medicine, one-third of the sudden deaths remain unexplained after medico-legal autopsy. A major proportion of these sudden unexplained deaths (SUD) are considered to be caused by inherited cardiac diseases. Sudden cardiac death (SCD) may be the first manifestation of these diseases. The purpose of this study was to explore the yield of next-generation sequencing of genes associated with SCD in a cohort of SUD victims. We investigated 100 genes associated with cardiac diseases in 61 young (1-50 years) SUD cases. DNA was captured with the Haloplex target enrichment system and sequenced using an Illumina MiSeq. The identified genetic variants were evaluated and classified as likely, unknown or unlikely to have a functional effect. The criteria for this classification were based on the literature, databases, conservation and prediction of the effect of the variant. We found that 21 (34%) individuals carried variants with a likely functional effect. Ten (40%) of these variants were located in genes associated with cardiomyopathies and 15 (60%) of the variants in genes associated with cardiac channelopathies. Nineteen individuals carried variants with unknown functional effect. Our findings indicate that broad genetic investigation of SUD victims increases the diagnostic outcome, and the investigation should comprise genes involved in both cardiomyopathies and cardiac channelopathies.

Genetic investigations of sudden unexpected deaths in infancy using next-generation sequencing of 100 genes associated with cardiac diseases.

Sudden infant death syndrome (SIDS) is the most frequent manner of post-perinatal death among infants. One of the suggested causes of the syndrome is inherited cardiac diseases, mainly channelopathies, that can trigger arrhythmias and sudden death. The purpose of this study was to investigate cases of sudden unexpected death in infancy (SUDI) for potential causative variants in 100 cardiac-associated genes. We investigated 47 SUDI cases of which 38 had previously been screened for variants in RYR2, KCNQ1, KCNH2 and SCN5A. Using the Haloplex Target Enrichment System (Agilent) and next-generation sequencing (NGS), the coding regions of 100 genes associated with inherited channelopathies and cardiomyopathies were captured and sequenced on the Illumina MiSeq platform. Sixteen (34%) of the SUDI cases had variants with likely functional effects, based on conservation, computational prediction and allele frequency, in one or more of the genes screened. The possible effects of the variants were not verified with family or functional studies. Eight (17%) of the SUDI cases had variants in genes affecting ion channel functions. The remaining eight cases had variants in genes associated with cardiomyopathies. In total, one third of the SUDI victims in a forensic setting had variants with likely functional effect that presumably contributed to the cause of death. The results support the assumption that channelopathies are important causes of SUDI. Thus, analysis of genes associated with cardiac diseases in SUDI victims is important in the forensic setting and a valuable supplement to the clinical investigation in all cases of sudden death.

Direct contact with endoderm-like cells efficiently induces cardiac progenitors from mouse and human pluripotent stem cells.

RATIONALE: Pluripotent stem cell-derived cardiac progenitor cells (CPCs) have emerged as a powerful tool to study cardiogenesis in vitro and a potential cell source for cardiac regenerative medicine. However, available methods to induce CPCs are not efficient or require high-cost cytokines with extensive optimization due to cell line variations. OBJECTIVE: Based on our in-vivo observation that early endodermal cells maintain contact with nascent pre-cardiac mesoderm, we hypothesized that direct physical contact with endoderm promotes induction of CPCs from pluripotent cells. METHOD AND RESULT: To test the hypothesis, we cocultured mouse embryonic stem (ES) cells with the endodermal cell line End2 by co-aggregation or End2-conditioned medium. Co-aggregation resulted in strong induction of Flk1(+) PDGFRa(+) CPCs in a dose-dependent manner, but the conditioned medium did not, indicating that direct contact is necessary for this process. To determine if direct contact with End2 cells also promotes the induction of committed cardiac progenitors, we utilized several mouse ES and induced pluripotent (iPS) cell lines expressing fluorescent proteins under regulation of the CPC lineage markers Nkx2.5 or Isl1. In agreement with earlier data, co-aggregation with End2 cells potently induces both Nkx2.5(+) and Isl1(+) CPCs, leading to a sheet of beating cardiomyocytes. Furthermore, co-aggregation with End2 cells greatly promotes the induction of KDR(+) PDGFRa(+) CPCs from human ES cells. CONCLUSIONS: Our co-aggregation method provides an efficient, simple and cost-effective way to induce CPCs from mouse and human pluripotent cells.