Allele-Specific Chromosome Removal after Cas9 Cleavage in Human Embryos.

Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.

CaMKII exacerbates heart failure progression by activating class I HDACs.

BACKGROUND: Persistent cardiac Ca2+/calmodulin dependent Kinase II (CaMKII) activation plays an essential role in heart failure development. However, the molecular mechanisms underlying CaMKII induced heart failure progression remains incompletely understood. Histone deacetylases (HDACs) are critical for transcriptional responses to stress, and contribute to expression of pathological genes causing adverse ventricular remodeling. Class I HDACs, including HDAC1, HDAC2 and HDAC3, promote pathological cardiac hypertrophy, whereas class IIa HDACs suppress cardiac hypertrophy. While it is known that CaMKII deactivates class IIa HDACs to enhance cardiac hypertrophy, the role of CaMKII in regulating class I HDACs during heart failure progression is unclear. METHODS AND RESULTS: CaMKII increases the deacetylase activity of recombinant HDAC1, HDAC2 and HDAC3 via in vitro phosphorylation assays. Phosphorylation sites on HDAC1 and HDAC3 are identified with mass spectrometry. HDAC1 activity is also increased in cardiac-specific CaMKIIδC transgenic mice (CaMKIIδC-tg). Beyond post-translational modifications, CaMKII induces HDAC1 and HDAC3 expression. HDAC1 and HDAC3 expression are significantly increased in CaMKIIδC-tg mice. Inhibition of CaMKII by overexpression of the inhibitory peptide AC3-I in the heart attenuates the upregulation of HDAC1 after myocardial infarction surgery. Importantly, a potent HDAC1 inhibitor Quisinostat improves downregulated autophagy genes and cardiac dysfunction in CaMKIIδC-tg mice. In addition to Quisinostat, selective class I HDACs inhibitors, Apicidin and Entinostat, HDAC3 specific inhibitor RGFP966, as well as HDAC1 and HDAC3 siRNA prevent CaMKII overexpression induced cardiac myocyte hypertrophy. CONCLUSION: CaMKII activates class I HDACs in heart failure, which may be a central mechanism for heart failure progression. Selective class I HDACs inhibition may be a novel therapeutic avenue to alleviate CaMKII hyperactivity induced cardiac dysfunction.

Myocardial brain-derived neurotrophic factor regulates cardiac bioenergetics through the transcription factor Yin Yang 1.

AIMS: Brain-derived neurotrophic factor (BDNF) is markedly decreased in heart failure patients. Both BDNF and its receptor, tropomyosin-related kinase receptor (TrkB), are expressed in cardiomyocytes; however, the role of myocardial BDNF signalling in cardiac pathophysiology is poorly understood. Here, we investigated the role of BDNF/TrkB signalling in cardiac stress response to exercise and pathological stress. METHODS AND RESULTS: We found that myocardial BDNF expression was increased in mice with swimming exercise but decreased in a mouse heart failure model and human failing hearts. Cardiac-specific TrkB knockout (cTrkB KO) mice displayed a blunted adaptive cardiac response to exercise, with attenuated upregulation of transcription factor networks controlling mitochondrial biogenesis/metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α). In response to pathological stress (transaortic constriction, TAC), cTrkB KO mice showed an exacerbated heart failure progression. The downregulation of PGC-1α in cTrkB KO mice exposed to exercise or TAC resulted in decreased cardiac energetics. We further unravelled that BDNF induces PGC-1α upregulation and bioenergetics through a novel signalling pathway, the pleiotropic transcription factor Yin Yang 1. CONCLUSION: Taken together, our findings suggest that myocardial BDNF plays a critical role in regulating cellular energetics in the cardiac stress response.

A novel test for annexin A5 M2 haplotyping in in vitro fertilization patients and preimplantation embryos.

OBJECTIVE: To develop a test for evaluating the annexin A5 M2 haplotype in in vitro fertilization patients and preimplantation embryos. DESIGN: Test performance was measured by comparing Sanger sequencing of parental blood DNA and quantitative real-time polymerase chain reaction (qPCR) of saliva DNA, 3 fibroblast cell line 7-cell aliquots and their corresponding purified DNA, 123 trophectoderm biopsy samples, and DNA isolated from 1 embryonic stem cell line along with the Mendelian inheritance expectations, embryo Sanger sequencing, and single-nucleotide polymorphism (SNP) microarray-based linkage analysis. SETTING: Preimplantation genetic testing laboratory research on IVF patient and embryo DNA. PATIENT(S): An assay was developed for the detection of the M2 haplotype on saliva samples of 6 in vitro fertilization patients. In addition, 13 patients who underwent preimplantation genetic testing with data on parental and embryo biopsy DNA available for research use were evaluated. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The concordance rates between Sanger sequencing, SNP array-based linkage analysis, and Mendelian inheritance expectations with qPCR. RESULT(S): The concordance rate between Sanger sequencing and qPCR was 100% on parental blood DNA and saliva DNA. The sample concordance rate between all replicates of 7-cell aliquots was 100%. The sample concordance rate between 3 cell lines used to prepare 7-cell aliquots and purified genomic DNA was 100%. The concordance rate between qPCR and Sanger sequencing results from a single trophectoderm biopsy and isolated embryonic stem cell line was 100%. The concordance rate of trophectoderm biopsy qPCR results and expectations from Mendelian inheritance rules was 97%; however, when SNP array-based linkage analysis was included, the concordance rate reached 100%. CONCLUSION(S): This study resulted in the development of a convenient saliva collection method and qPCR-based genotyping method to screen for the M2 haplotype. In addition, a novel method for testing preimplantation embryos has been established, providing an alternative to the use of low molecular weight heparin, through selection of embryos without the M2 haplotype.

Utility and First Clinical Application of Screening Embryos for Polygenic Disease Risk Reduction.

For over 2 decades preimplantation genetic testing (PGT) has been in clinical use to reduce the risk of miscarriage and genetic disease in patients with advanced maternal age and risk of transmitting disease. Recently developed methods of genome-wide genotyping and machine learning algorithms now offer the ability to genotype embryos for polygenic disease risk with accuracy equivalent to adults. In addition, contemporary studies on adults indicate the ability to predict polygenic disorders with risk equivalent to monogenic disorders. Existing biobanks provide opportunities to model the clinical utility of polygenic disease risk reduction among sibling adults. Here, we provide a mathematical model for the use of embryo screening to reduce the risk of type 1 diabetes. Results indicate a 45-72% reduced risk with blinded genetic selection of one sibling. The first clinical case of polygenic risk scoring in human preimplantation embryos from patients with a family history of complex disease is reported. In addition to these data, several common and accepted practices place PGT for polygenic disease risk in the applicable context of contemporary reproductive medicine. In addition, prediction of risk for PCOS, endometriosis, and aneuploidy are of particular interest and relevance to patients with infertility and represent an important focus of future research on polygenic risk scoring in embryos.

Validation of concurrent preimplantation genetic testing for polygenic and monogenic disorders, structural rearrangements, and whole and segmental chromosome aneuploidy with a single universal platform.

Preimplantation genetic testing (PGT) has been successfully applied to reduce the risk of miscarriage, improve IVF success rates, and prevent inheritance of monogenic disease and unbalanced translocations. The present study provides the first method capable of simultaneous testing of aneuploidy (PGT-A), structural rearrangements (PGT-SR), and monogenic (PGT-M) disorders using a single platform. Using positive controls to establish performance characteristics, accuracies of 97 to >99% for each type of testing were observed. In addition, this study expands PGT to include predicting the risk of polygenic disorders (PGT-P) for the first time. Performance was established for two common diseases, hypothyroidism and type 1 diabetes, based upon availability of positive control samples from commercially available repositories. Data from the UK Biobank, eMERGE, and T1DBASE were used to establish and validate SNP-based predictors of each disease (7,311 SNPs for hypothyroidism and 82 for type 1 diabetes). Area under the curve of disease status prediction from genotypes alone were 0.71 for hypothyroidism and 0.68 for type 1 diabetes. The availability of expanded PGT to evaluate the risk of polygenic disorders in the preimplantation embryo has the potential to lower the prevalence of common genetic disease in humans.

Bilateral acute postoperative retinal detachment after cataract extraction: case report and review of the literature.

A 57-year-old white man had extracapsular cataract extraction complicated by vitreous loss. On postoperative day 1, he was noted to have a total retinal detachment (RD) with vitreous hemorrhage. No predisposing anatomic risk factors were present except for the vitreous loss. During the RD repair, 2 small superior tears were discovered. Eleven months later, the patient had uneventful phacoemulsification in the fellow eye. On postoperative day 1, he again had a total RD with a superior retinal tear. Meticulous retinal evaluation had been performed preoperatively, and no holes or tears were discovered. The RD was repaired, and the best corrected visual acuity at the last examination was 20/40 in both eyes.

Support for the homeobox transcription factor gene ENGRAILED 2 as an autism spectrum disorder susceptibility locus.

Our previous research involving 167 nuclear families from the Autism Genetic Resource Exchange (AGRE) demonstrated that two intronic SNPs, rs1861972 and rs1861973, in the homeodomain transcription factor gene ENGRAILED 2 (EN2) are significantly associated with autism spectrum disorder (ASD). In this study, significant replication of association for rs1861972 and rs1861973 is reported for two additional data sets: an independent set of 222 AGRE families (rs1861972-rs1861973 haplotype, P=.0016) and a separate sample of 129 National Institutes of Mental Health families (rs1861972-rs1861973 haplotype, P=.0431). Association analysis of the haplotype in the combined sample of both AGRE data sets (389 families) produced a P value of .0000033, whereas combining all three data sets (518 families) produced a P value of .00000035. Population-attributable risk calculations for the associated haplotype, performed using the entire sample of 518 families, determined that the risk allele contributes to as many as 40% of ASD cases in the general population. Linkage disequilibrium (LD) mapping with the use of polymorphisms distributed throughout the gene has shown that only intronic SNPs are in strong LD with rs1861972 and rs1861973. Resequencing and association analysis of all intronic SNPs have identified alleles associated with ASD, which makes them candidates for future functional analysis. Finally, to begin defining the function of EN2 during development, mouse En2 was ectopically expressed in cortical precursors. Fewer En2-transfected cells than controls displayed a differentiated phenotype. Together, these data provide further genetic evidence that EN2 might act as an ASD susceptibility locus, and they suggest that a risk allele that perturbs the spatial/temporal expression of EN2 could significantly alter normal brain development.