AMFR dysfunction causes autosomal recessive spastic paraplegia in human that is amenable to statin treatment in a preclinical model.

Hereditary spastic paraplegias (HSP) are rare, inherited neurodegenerative or neurodevelopmental disorders that mainly present with lower limb spasticity and muscle weakness due to motor neuron dysfunction. Whole genome sequencing identified bi-allelic truncating variants in AMFR, encoding a RING-H2 finger E3 ubiquitin ligase anchored at the membrane of the endoplasmic reticulum (ER), in two previously genetically unexplained HSP-affected siblings. Subsequently, international collaboration recognized additional HSP-affected individuals with similar bi-allelic truncating AMFR variants, resulting in a cohort of 20 individuals from 8 unrelated, consanguineous families. Variants segregated with a phenotype of mainly pure but also complex HSP consisting of global developmental delay, mild intellectual disability, motor dysfunction, and progressive spasticity. Patient-derived fibroblasts, neural stem cells (NSCs), and in vivo zebrafish modeling were used to investigate pathomechanisms, including initial preclinical therapy assessment. The absence of AMFR disturbs lipid homeostasis, causing lipid droplet accumulation in NSCs and patient-derived fibroblasts which is rescued upon AMFR re-expression. Electron microscopy indicates ER morphology alterations in the absence of AMFR. Similar findings are seen in amfra-/- zebrafish larvae, in addition to altered touch-evoked escape response and defects in motor neuron branching, phenocopying the HSP observed in patients. Interestingly, administration of FDA-approved statins improves touch-evoked escape response and motor neuron branching defects in amfra-/- zebrafish larvae, suggesting potential therapeutic implications. Our genetic and functional studies identify bi-allelic truncating variants in AMFR as a cause of a novel autosomal recessive HSP by altering lipid metabolism, which may potentially be therapeutically modulated using precision medicine with statins.

Comprehensive multi-omics integration identifies differentially active enhancers during human brain development with clinical relevance.

BACKGROUND: Non-coding regulatory elements (NCREs), such as enhancers, play a crucial role in gene regulation, and genetic aberrations in NCREs can lead to human disease, including brain disorders. The human brain is a complex organ that is susceptible to numerous disorders; many of these are caused by genetic changes, but a multitude remain currently unexplained. Understanding NCREs acting during brain development has the potential to shed light on previously unrecognized genetic causes of human brain disease. Despite immense community-wide efforts to understand the role of the non-coding genome and NCREs, annotating functional NCREs remains challenging. METHODS: Here we performed an integrative computational analysis of virtually all currently available epigenome data sets related to human fetal brain. RESULTS: Our in-depth analysis unravels 39,709 differentially active enhancers (DAEs) that show dynamic epigenomic rearrangement during early stages of human brain development, indicating likely biological function. Many of these DAEs are linked to clinically relevant genes, and functional validation of selected DAEs in cell models and zebrafish confirms their role in gene regulation. Compared to enhancers without dynamic epigenomic rearrangement, DAEs are subjected to higher sequence constraints in humans, have distinct sequence characteristics and are bound by a distinct transcription factor landscape. DAEs are enriched for GWAS loci for brain-related traits and for genetic variation found in individuals with neurodevelopmental disorders, including autism. CONCLUSION: This compendium of high-confidence enhancers will assist in deciphering the mechanism behind developmental genetics of human brain and will be relevant to uncover missing heritability in human genetic brain disorders.

Case Report: Two New Cases of Chromosome 12q14 Deletions and Review of the Literature.

Interstitial deletions on the long arm of chromosome 12 (12q deletions) are rare, and are associated with intellectual disability, developmental delay, failure to thrive and congenital anomalies. The precise genotype-phenotype correlations of different deletions has not been completely resolved. Ascertaining individuals with overlapping deletions and complex phenotypes may help to identify causative genes and improve understanding of 12q deletion syndromes. We here describe two individuals with non-overlapping 12q14 deletions encountered at our clinical genetics outpatient clinic and perform a review of all previously published interstitial 12q deletions to further delineate genotype-phenotype correlations. Both individuals presented with a neurodevelopmental disorder with various degrees of intellectual disability, failure to thrive and dysmorphic features. Previously, larger deletions overlapping large parts of the deletions encountered in both individuals have been described. Whereas, individual 1 seems to fit into the previously described phenotypic spectrum of the 12q14 microdeletion syndrome, individual 2 displays more severe neurological symptoms, which are likely caused by haploinsufficiency of the BAF complex member SMARCC2, which is included in the deletion. We furthermore perform a review of all previously published interstitial 12q deletions which we found to cluster amongst 5 regions on chromosome 12, to further delineate genotype-phenotype correlations, and we discuss likely disease relevant genes for each of these deletion clusters. Together, this expands knowledge on deletions on chromosome 12q which might facilitate patient counseling. Also, it illustrates that re-analysis of previously described microdeletions syndromes in the next generation sequencing era can be useful to delineate genotype-phenotype correlations and identify disease relevant genes in individuals with neurodevelopmental disorders.

RNA-seq analysis of different inflammatory reactions induced by lipopolysaccharide and lipoteichoic acid in bovine mammary epithelial cells.

Lipopolysaccharide (LPS) and lipoteichoic acid (LTA) are key virulence factors of Escherichia coli and Staphylococcus aureus respectively, and both of them could cause inflammatory reaction in bovine mammary glands. In this study, we used bovine mammary epithelial cells (BMECs) as pattern recognition receptors and stimulated them with LPS or LTA to investigate the global transcriptional response variations of BMECs to these two different virulent factors through RNA-Seq analysis. We found 100 differentially expressed genes (DEGs) with 95 up-regulated and 5 down-regulated genes in LPS-treated group, whereas 24 DEGs with 12 up-regulated and 12 down-regulated genes in LTA-treated group compared to control. Although the number and expression changes of DEGs are significantly different between LPS vs Control and LTA vs Control, KEGG pathway enrichment analysis showed the majorities of DEGs in each pair were enriched on cytokine-cytokine receptor interaction, NF-κB signaling pathway, and NOD-like receptor signaling pathway, especially cytokines and chemokines. These results provided a comprehensive analysis of gene expression profiles elicited by LPS and LTA in BMECs, contributing to the understanding of early "pathogen-host" interactions during intramammary infections.

Identification and characterization of ERV transcripts in goat embryos.

Endogenous retroviruses (ERVs), which are abundant in mammalian genomes, can modulate the expression of nearby genes, and their expression is dynamic and stage-specific during early embryonic development in mice and humans. However, the functions and mechanisms of ERV elements in regulating embryonic development remain unclear. Here, we utilized several methods to determine the contribution of ERVs to the makeup and regulation of transcripts during embryonic genome activation (EGA). We constructed an ERV library and embryo RNA-seq library (IVF_2c and IVF_8c) of goat to serve as our research basis. The GO and KEGG analysis of nearby ERV genes revealed that some ERV elements may be associated with embryonic development. RNA-seq results were consistent with the features of EGA. To obtain the transcripts derived from the ERV sequences, we blasted the ERV sequences with embryonic transcripts and identified three lncRNAs and one mRNA that were highly expressed in IVF-8c rather than in IVF-2c (q-value <0.05). Then, we validated the expression patterns of nine ERV-related transcripts during early developmental stages and knocked down three high-expression transcripts in EGA. The knockdown of lncRNA TCONS_00460156 or mRNA HSD17B11 significantly decreased the developmental rate of IVF embryos. Our findings suggested that some transcripts from ERVs are essential for the early embryonic development of goat, and analyzing the ERV expression profile during goat EGA may help elucidate the molecular mechanisms of ERV in regulating embryonic development.

Overexpression of Tet3 in donor cells enhances goat somatic cell nuclear transfer efficiency.

Ten-eleven translocation 3 (TET3) mediates active DNA demethylation of paternal genomes during mouse embryonic development. However, the mechanism of DNA demethylation in goat embryos remains unknown. In addition, aberrant DNA methylation reprogramming prevalently occurs in embryos cloned by somatic cell nuclear transfer (SCNT). In this study, we reported that TET3 is a key factor in DNA demethylation in goat pre-implantation embryos. Knockdown of Tet3 hindered DNA demethylation at the two- to four-cell stage in goat embryos and decreased Nanog expression in blastocysts. Overexpression of Tet3 in somatic cells can initiate DNA demethylation, reduce 5-methylcytosine level, increase 5-hydroxymethylcytosine level and promote the expression of key pluripotency genes. After SCNT, overexpression of Tet3 in donor cells corrected abnormal DNA hypermethylation of cloned embryos and significantly enhanced in vitro and in vivo developmental rate (P < 0.05). We conclude that overexpression of Tet3 in donor cells significantly improves goat SCNT efficiency.

Treating donor cells with 2-PCPA corrects aberrant histone H3K4 dimethylation and improves cloned goat embryo development.

Epigenetic modifications extensively occur in mammalian embryonic development and cell differentiation process. They play an essential role in the reprogramming of nuclei during somatic cell nuclear transfer (SCNT) and subsequent in vitro embryonic development. Recently, SCNT embryos have been verified to contain a subnormal level of histone H3K4 dimethylation (H3K4me2) in contrast to in vitro fertilized embryos. This finding suggested that increasing H3K4me2 levels may ameliorate the aberrant development of cloned embryos. In this study, we investigated the influence of treating donor cells with trans-2-Phenylcyclopropylamine (2-PCPA), a specific inhibitor of lysine-specific demethylase 1 (LSD1), on embryogenesis, H3K4me2 level, and gene expression in cloned goat embryos. Treated goat fetal fibroblast cells (GFFs) with 2-PCPA served as donor cells for subsequent SCNT. Results showed that H3K4me2 levels in treated GFFs increased gradually with the increasing 2-PCPA concentration (p < 0.05) and had no obvious influence in cell viability. The 2-PCPA-induced up-regulation of H3K4me2 levels led to G0/G1 cell cycle arrest and the difference was significant at 2µM compared with the control group (p < 0.05). Interestingly, the development rate of goat SCNT embryos in vitro was significantly improved and aberrant H3K4me2 levels were effectively corrected in 2-PCPA-treated SCNT embryos in contrast to that in SCNT control embryos. Moreover, 2-PCPA treatment promoted the mRNA expression of key developmental genes Oct4 and Sox2 (p < 0.05) without affecting the expression levels of imprinted genes IGF2R and H19 in goat SCNT embryos. These results indicated that abnormal H3K4me2 status can be corrected and SCNT embryo development can be promoted through treatment of donor cells with 2-PCPA. ABBREVIATIONS: SCNT: somatic cell nuclear transfer; H3K4me2: H3K4 dimethylation; 2-PCPA: trans-2-Phenylcyclopropylamine; LSD1: lysine-specific demethylase 1; GFFs: goat fetal fibroblast cells; IVF: in vitro fertilization; iPS: induced pluripotent stem; PBS: phosphate-buffered saline; IVM: in vitro maturation; RNAPII: RNA polymerase II; HMTs: histone methyltransferase.

Protective Effects of Quercetin Against Cadmium Chloride-Induced Oxidative Injury in Goat Sperm and Zygotes.

Quercetin, a plant-derived flavonoid, is frequently used as an antioxidant for efficient anti-oxidative capacity. However, whether quercetin has protective effects on goat sperm and preimplantation embryos against Cd2+-induced oxidative injury is still unclear. So, we researched the influence of quercetin on goat sperm and zygotes respectively under the oxidative stress induced by Cd2+. In our study, quercetin decreased the malonaldehyde (MDA) and reactive oxygen species (ROS) levels caused by Cd2+ in goat sperm (p < 0.05), which facilitated sperm characteristics including motility, survival rates, membrane integrity, and mitochondria activity during storage in vitro and subsequent embryo development (p < 0.05). Moreover, in goat zygotes, quercetin decreased peroxidation products including ROS, MDA, and carbonyl through preserving or maintaining mitochondrial function, gene expression, and anti-oxidative products such as glutathione peroxidase, superoxide dismutase, and catalase, which ameliorated subsequent embryo development and embryo quality (p < 0.05). Taken together, these results suggest that quercetin protects both goat sperm and preimplantation embryos from Cd2+-induced oxidative stress.