OBJECTIVE: Retrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos.
METHODS: Sixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls.
RESULTS: Twenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos.
CONCLUSIONS: Several studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.

Haplotype information is crucial for biomedical and population genetics research. However, current strategies to produce de-novo haplotype-resolved assemblies often require either difficult-to-acquire parental data or an intermediate haplotype-collapsed assembly. Here, we present Graphasing, a workflow which synthesizes the global phase signal of Strand-seq with assembly graph topology to produce chromosome-scale de-novo haplotypes for diploid genomes. Graphasing readily integrates with any assembly workflow that both outputs an assembly graph and has a haplotype assembly mode. Graphasing performs comparably to trio-phasing in contiguity, phasing accuracy, and assembly quality, outperforms Hi-C in phasing accuracy, and generates human assemblies with over 18 chromosome-spanning haplotypes.

Balancing relative expression of pathway genes to minimize flux bottlenecks and metabolic burden is one of the key challenges in metabolic engineering. This is especially relevant for iterative pathways, such as reverse β-oxidation (rBOX) pathway, which require control of flux partition at multiple nodes to achieve efficient synthesis of target products. Here, we develop a plasmid-based inducible system for orthogonal control of gene expression (referred to as the TriO system) and demonstrate its utility in the rBOX pathway. Leveraging effortless construction of TriO vectors in a plug-and-play manner, we simultaneously explored the solution space for enzyme choice and relative expression levels. Remarkably, varying individual expression levels led to substantial change in product specificity ranging from no production to optimal performance of about 90% of the theoretical yield of the desired products. We obtained titers of 6.3 g/L butyrate, 2.2 g/L butanol and 4.0 g/L hexanoate from glycerol in E. coli, which exceed the best titers previously reported using equivalent enzyme combinations. Since a similar system behavior was observed with alternative termination routes and higher-order iterations, we envision our approach to be broadly applicable to other iterative pathways besides the rBOX. Considering that high throughput, automated strain construction using combinatorial promoter and RBS libraries remain out of reach for many researchers, especially in academia, tools like the TriO system could democratize the testing and evaluation of pathway designs by reducing cost, time and infrastructure requirements.

The RhoGEF Trio is a large multi-domain protein and an activator of the small GTPases Rac1, RhoG, and RhoA. Although Trio has been implicated in many cellular mechanisms like leukocyte transendothelial migration, cell-cell junction stability, lamellipodia formation, axon outgrowth, and muscle fusion, it remains unclear how Trio is activated. Using stable isotope labelling by amino acids in cell culture (SILAC)-based mass spectrometry analysis of endothelial cells, we identified two serine residues (S1785/S1786) located in between the two exchange domains of Trio that were highly phosphorylated upon short thrombin treatment. Using phosphomimetic Trio S1785D/S1786D double mutants, we did not find an increase in Rac1/RhoG activity, indicating that the phosphorylation events do not increase Trio exchange activity. However, we found that the Trio mutants localized more strongly at cell-cell junctions and prevented junction destabilization upon thrombin treatment, judged by junction linearity. Our data suggest that serine phosphorylation of Trio potentiates the localization of Trio to junctional regions, resulting in locally promoting the exchange for Rac1 at junction regions and increasing endothelial cell-cell junction stability upon permeability-inducing reagents such as thrombin.

Single nucleotide polymorphism (SNP) chromosome microarray is well established for investigation of children with intellectual deficit/development delay and prenatal diagnosis of fetal malformation but has also emerged for uniparental disomy (UPD) genotyping. Despite published guidelines on clinical indications for testing there are no laboratory guidelines published for performing SNP microarray UPD genotyping. We evaluated SNP microarray UPD genotyping using Illumina beadchips on family trios/duos within a clinical cohort (n=98) and then explored our findings in a post-study audit (n=123). UPD occurred in 18.6% and 19.5% cases, respectively, with chromosome 15 most frequent (62.5% and 25.0%). UPD was predominantly maternal in origin (87.5% and 79.2%), highest in suspected genomic imprinting disorder cases (56.3% and 41.7%) but absent amongst children of translocation carriers. We assessed regions of homozygosity among UPD cases. The smallest interstitial and terminal regions were 2.5 Mb and 9.3 Mb, respectively. We found regions of homozygosity confounded genotyping in a consanguineous case with UPD15 and another with segmental UPD due to non-informative probes. In a unique case with chromosome 15q UPD mosaicism, we established the detection limit of mosaicism as ∼5%. From the benefits and pitfalls identified in this study, we propose a testing model and recommendations for UPD genotyping by SNP microarray.

Intellectual disability with developmental delay is the most common developmental disorder. However, this diagnosis is rarely associated with congenital cardiomyopathy. In the current report, we present the case of a patient suffering from dilated cardiomyopathy and developmental delay.
Neurological pathology in a newborn was diagnosed immediately after birth, and the acquisition of psychomotor skills lagged behind by 3-4 months during the first year of life. WES analysis of the proband did not reveal a causal variant, so the search was extended to trio.
Trio sequencing revealed a de novo missense variant in the CAMK2D gene (p.Arg275His), that is, according to the OMIM database and available literature, not currently associated with any specific inborn disease. The expression of Ca2+/calmodulin-dependent protein kinase II delta (CaMKIIδ) protein is known to be increased in the heart tissues from patients with dilated cardiomyopathy. The functional effect of the CaMKIIδ Arg275His mutant was recently reported; however, no specific mechanism of its pathogenicity was proposed. A structural analysis and comparison of available three-dimensional structures of CaMKIIδ confirmed the probable pathogenicity of the observed missense variant.
We suggest that the CaMKIIδ Arg275His variant is highly likely the cause of dilated cardiomyopathy and neurodevelopmental disorders.

OBJECTIVE: To assess the efficacy of raltegravir, etravirine and darunavir/ritonavir (TRIO regimen) in treatment-experienced patients with human immunodeficiency virus-1 (HIV-1) infection by describing the proportion of patients who experienced virological failure (VF) at Week 24. The secondary objectives were to assess the HIV-1 plasma viral load (pVL) after Week 24, the proportion of patients who were receiving dual therapy or monotherapy at the last visit, and the number of deaths.
METHODS: Patients from the ANRS CO3 Aquitaine Cohort who were prescribed the TRIO regimen between February 2007 and September 2018 were classified into two groups based on their pVL at study inclusion: the virological failure group (VFG; pVL >50 copies/mL) and the virologically suppressed group (VSG; pVL <50 copies/mL). The impact of baseline pVL and genotypic susceptibility score (GSS) on VF was analysed.
RESULTS: In total, 184 patients were enrolled in this study, with 123 (66.8%) in the VFG and 61 (33.2%) in the VSG. The median length of follow-up was 7.5 (interquartile range 4.1-9.6) years, and 29 (15.8%) patients died. Thirty-seven (25.5%) patients experienced VF at Week 24, including 32/145 (32.7%) in the VFG and 5/47 (10.6%) in the VSG (P<0.01). Resistance-associated mutations were detected in integrase, reverse transcriptase and protease for 7/37 (18.9%), 3/37 (8.1%) and 1/37 (2.7%) patients, respectively. High pVL and GSS at baseline were independently associated with VF. At the last visit, 76/184 (41.3%) patients were still receiving the TRIO regimen, while 55/184 (29.9%) were receiving dual therapy and 1/184 (0.5%) was receiving protease inhibitor monotherapy. Among the 56 patients receiving dual therapy or monotherapy, 51 (96.2%) had pVL <50 copies/mL.
CONCLUSIONS: Despite a high level of mutation resistance at baseline, long-term virological follow-up was favourable and one-third of patients were eligible for drug-reducing strategies.

Syndromic intellectual disability (ID) with accompanying primary microcephaly is a group of rare neurodevelopmental disorders exhibiting extreme genetic and clinical heterogeneity. This layered heterogeneity can partially be resolved by unbiased genetic approaches targeting the genome with next generation sequencing (NGS) technologies, including exome sequencing (ES).
This study was performed to dissect the clinical and genetic features in five distinct IDM cases.
Singleton or trio ES approach followed by in-depth variant analysis using alternative inheritance models was performed.
We have identified biallelic loss of function variants in genes WDR62 and AP4M1 in three families, together with de novo missense variants in genes SOX11 and TRIO in two families. ES based haplotype analysis in two cases upon identification of an identical WDR62 variant in the homozygous state in two cases was suggestive of a small shared haplotype of 0.1 Mb. Additionally, we have shown a paternal origin for the de novo variant in TRIO via a polymorphic tag SNP, which enlightens the mutational mechanism for this variant.
In populations with high parental consanguinity, an autosomal recessive inheritance pattern for data analysis is usually the most obvious choice. Therefore, heterozygous variants may be overlooked in standard NGS analyses in consanguineous families. Our findings underlie the importance of using multiple inheritance models in NGS data analysis.

Compelling evidence indicates that in Huntington\'s disease (HD), mutation of huntingtin (HTT) alters several aspects of early brain development such as synaptogenesis. It is not clear to what extent the partial loss of wild-type HTT function contributes to these abnormalities. Here we investigate the function of HTT in the formation of spines. Although larger spines normally correlate with more synaptic activity, cell-autonomous depletion of HTT leads to enlarged spines but reduced excitatory synaptic function. We find that HTT is required for the proper turnover of endogenous actin and to recruit AMPA receptors at active synapses; loss of HTT leads to LIM kinase (LIMK) hyperactivation, which maintains cofilin in its inactive state. HTT therefore influences actin dynamics through the LIMK-cofilin pathway. Loss of HTT uncouples spine structure from synaptic function, which may contribute to the ultimate development of HD symptoms.

Germline or constitutional chromoanagenesis-related complex chromosomal rearrangements (CCRs) are rare, apparently \"all-at-once\", catastrophic events that occur in a single cell cycle, exhibit an unexpected complexity, and sometimes correlate with a severe abnormal phenotype. The term chromoanagenesis encompasses three distinct phenomena, namely chromothripsis, chromoanasynthesis, and chromoplexy. Herein, we found hallmarks of chromothripsis and chromoplexy in an ultra-complex t(5;7;21)dn involving several disordered breakpoint junctions (BPJs) accompanied by some microdeletions and the disruption of neurodevelopmental genes in a patient with a phenotype resembling autosomal dominant MRD44 (OMIM 617061). G-banded chromosomes and FISH showed that the CCR implied the translocation of the 5p15.2→pter segment onto 7q11.23; in turn, the fragment 7q11.23→qter of der(7) separated into two pieces: the segment q11.23→q32 translocated onto 5p15.2 and fused to 21q22.1→ter in the der(5) while the distal 7q32→qter segment translocated onto der(21) at q22.1. Subsequent whole-genome sequencing unveiled that CCT5, CMBL, RETREG1, MYO10, and TRIO from der(5), IMMP2L, TES, VPS37D, DUS4L, TYW1B, and FEZF1-AS1 from der(7), and TIAM1 and SOD1 from der(21), were disrupted by BPJs, whereas some other genes (predicted to be haplosufficient or inconsequential) were completely deleted. Although remarkably CCT5, TRIO, TES, MYO10, and TIAM1 (and even VPS37D) cooperate in key biological processes for normal neuronal development such as cell adhesion, migration, growth, and/or cytoskeleton formation, the disruption of TRIO most likely caused the patient\'s MRD44-like phenotype, including intellectual disability, microcephaly, finger anomalies, and facial dysmorphia. Our observation represents the first truncation of TRIO related to a chromoanagenesis event and therefore expands the mutational spectrum of this crucial gene. Moreover, our findings indicate that more than one mechanism is involved in modeling the architecture of ultra-complex rearrangements.