The overall understanding of the molecular etiologies of intellectual disability (ID) and developmental delay (DD) is increasing as next-generation sequencing technologies identify genetic variants in individuals with such disorders. However, detailed analyses conclusively confirming these variants, as well as the underlying molecular mechanisms explaining the diseases, are often lacking. Here, we report on an ID syndrome caused by de novo heterozygous loss-of-function (LoF) mutations in SON. The syndrome is characterized by ID and/or DD, malformations of the cerebral cortex, epilepsy, vision problems, musculoskeletal abnormalities, and congenital malformations. Knockdown of son in zebrafish resulted in severe malformation of the spine, brain, and eyes. Importantly, analyses of RNA from affected individuals revealed that genes critical for neuronal migration and cortex organization (TUBG1, FLNA, PNKP, WDR62, PSMD3, and HDAC6) and metabolism (PCK2, PFKL, IDH2, ACY1, and ADA) are significantly downregulated because of the accumulation of mis-spliced transcripts resulting from erroneous SON-mediated RNA splicing. Our data highlight SON as a master regulator governing neurodevelopment and demonstrate the importance of SON-mediated RNA splicing in human development.

In humans, Mammalian Target of Rapamycin (MTOR) encodes a 300 kDa serine/ threonine protein kinase that is ubiquitously expressed, particularly at high levels in brain. MTOR functions as an integrator of multiple cellular processes, and in so doing either directly or indirectly regulates the phosphorylation of at least 800 proteins. While somatic MTOR mutations have been recognized in tumors for many years, and more recently in hemimegalencephaly, germline MTOR mutations have rarely been described.

Foxtail millet (Setaria italica (L.) P. Beauv), which belongs to the Panicoideae tribe of the Poaceae, is an important grain crop widely grown in Northern China and India. It is currently developing into a novel model species for functional genomics of the Panicoideae as a result of its fully available reference genome sequence, small diploid genome (2n=18, ~510Mb), short life cycle, small stature and prolific seed production. Argonaute 1 (AGO1), belonging to the argonaute (AGO) protein family, recruits small RNAs and regulates plant growth and development. Here, we characterized an AGO1 mutant (siago1b) in foxtail millet, which was induced by ethyl methanesulfonate treatment. The mutant exhibited pleiotropic developmental defects, including dwarfing stem, narrow and rolled leaves, smaller panicles and lower rates of seed setting. Map-based cloning analysis demonstrated that these phenotypic variations were attributed to a C-A transversion, and a 7-bp deletion in the C-terminus of the SiAGO1b gene in siago1b Yeast two-hybrid assays and BiFC experiments revealed that the mutated region was an essential functional motif for the interaction between SiAGO1b and SiHYL1. Furthermore, 1598 differentially expressed genes were detected via RNA-seq-based comparison of SiAGO1b and wild-type plants, which revealed that SiAGO1b mutation influenced multiple biological processes, including energy metabolism, cell growth, programmed death and abiotic stress responses in foxtail millet. This study may provide a better understanding of the mechanisms by which SiAGO1b regulates the growth and development of crops.

Foxtail millet (Setaria italica) is a diploid C4 panicoid species. Because of its prominent drought resistance, small genome size, self-pollination, and short life cycle, foxtail millet has become an ideal model system for studying drought tolerance of crops. MicroRNAs (miRNAs) are endogenous, small RNAs that play important regulatory roles in the development and stress response in plants.

Hematopoietic stem cells (HSCs) underlie the production of blood and immune cells for the lifetime of an organism. In vertebrate embryos, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in vitro from pluripotent precursors, partly because the full complement of required signaling inputs remains to be determined. Here, we show that TNFR2 via TNF? activates the Notch and NF-?B signaling pathways to establish HSC fate, indicating a requirement for inflammatory signaling in HSC generation. We determine that primitive neutrophils are the major source of TNF?, assigning a role for transient innate immune cells in establishing the HSC program. These results demonstrate that proinflammatory signaling, in the absence of infection, is utilized by the developing embryo to generate the lineal precursors of the adult hematopoietic system.

Haematopoietic stem cells (HSCs) derive from haemogenic endothelial cells of the primitive dorsal aorta (DA) during vertebrate embryogenesis. The molecular mechanisms governing this unique endothelial to haematopoietic transition remain unclear. Here, we demonstrate a novel requirement for fibroblast growth factor (FGF) signalling in HSC emergence. This requirement is non-cell-autonomous, and acts within the somite to bridge the Wnt and Notch signalling pathways. We previously demonstrated that Wnt16 regulates the somitic expression of two Notch ligands, deltaC (dlc) and deltaD (dld), whose combined function is required for HSC fate. How Wnt16 connects to Notch function has remained an open question. Our current studies demonstrate that FGF signalling, via FGF receptor 4 (Fgfr4), mediates a signal-transduction pathway between Wnt16 and Dlc, but not Dld, to regulate HSC specification. Our findings demonstrate that FGF signalling acts as a key molecular relay within the developmental HSC niche to instruct HSC fate.

To assess the efficacy of rituximab in treatment of refractory nephrotic syndrome (NS) compared with other agents.Studies were searched from Web of Science, PubMed, and CNKI up to April 2016. The standardized mean difference or relative risk or odds ratio and 95% confidence intervals were used to assess the efficacy of rituximab treatment compared with other agents in refractory NS.Totally, 8 studies were included. The present study showed that there was a significant higher relapse-free survival rate in rituximab group than that in the other agents group. Compared with other agents, rituximab did not significantly improve the complete and overall remission rate, serum albumin levels. Rituximab also did not decrease the serum creatinine, urinary protein, and serum cholesterol levels. However, compared with other agents, the adult patients had a higher serum cholesterol levels after treatment with rituximab.Rituximab promised to be a new agent in the treatment of refractory NS; it also could be used as an alternative to conventional immunosuppressive drugs-dependent or drugs-resistant. However, more high-quality, large sample, and multicenter randomized controlled trials are needed to further confirm the efficacy of rituximab in treatment of refractory NS.

Neurodevelopment is a transcriptionally orchestrated process. Cyclin K, a regulator of transcription encoded by CCNK, is thought to play a critical role in the RNA polymerase II-mediated activities. However, dysfunction of CCNK has not been linked to genetic disorders. In this study, we identified three unrelated individuals harboring de novo heterozygous copy number loss of CCNK in an overlapping 14q32.3 region and one individual harboring a de novo nonsynonymous variant c.331A>G (p.Lys111Glu) in CCNK. These four individuals, though from different ethnic backgrounds, shared a common phenotype of developmental delay and intellectual disability (DD/ID), language defects, and distinctive facial dysmorphism including high hairline, hypertelorism, thin eyebrows, dysmorphic ears, broad nasal bridge and tip, and narrow jaw. Functional assay in zebrafish larvae showed that Ccnk knockdown resulted in defective brain development, small eyes, and curly spinal cord. These defects were partially rescued by wild-type mRNA coding CCNK but not the mRNA with the identified likely pathogenic variant c.331A>G, supporting a causal role of CCNK variants in neurodevelopmental disorders. Taken together, we reported a syndromic neurodevelopmental disorder with DD/ID and facial characteristics caused by CCNK variations, possibly through a mechanism of haploinsufficiency.

Whole exome sequencing using a parent-child trio design to identify de novo mutations provides an efficient method to identify novel genes for rare diseases with low reproductive fitness that are difficult to study by more classical genetic methods of linkage analysis. We describe a 15 y old female with severe static encephalopathy, intellectual disability, and generalized epilepsy. After extensive metabolic and genetic testing, whole exome sequencing identified a novel de novo variant in Synaptosomal-associated protein-25 (SNAP25), c.142G > T p.Phe48Val alteration. This variant is predicted to be damaging by all prediction algorithms. SNAP25 is part of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complex which is involved in exocytotic release of neurotransmitters. Genetic alterations in Snap25 in animal models can cause anxiety-related behavior, ataxia and seizures. We suggest that SNAP25 mutations in humans are a novel genetic cause of intellectual disability and epilepsy.

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium.

Protein-coding genes are considered to be a dominant component of the eukaryotic transcriptome; however, many studies have shown that intergenic, non-coding transcripts also play an important role. Long intergenic non-coding RNAs (lincRNAs) were found to play a vital role in human and Arabidopsis. However, lincRNAs and their regulatory roles remain poorly characterized in woody plants, especially Populus trichocarpa (P. trichocarpa). A large set of Populus RNA-Seq data were examined with high sequencing depth under control and drought conditions and a total of 2542 lincRNA candidates were identified. In total, 51 lincRNAs and 20 lincRNAs were identified as putative targets and target mimics of known Populus miRNAs, respectively. A total of 504 lincRNAs were found to be drought responsive, eight of which were confirmed by RT-qPCR. These findings provide a comprehensive view of Populus lincRNAs, which will enable in-depth functional analysis.

To evaluate the phenotypic spectrum caused by mutations in dynamin 1 (DNM1), encoding the presynaptic protein DNM1, and to investigate possible genotype-phenotype correlations and predicted functional consequences based on structural modeling.

Fucosyltransferase 8 (FUT8) encodes a Golgi-localized α1,6 fucosyltransferase that is essential for transferring the monosaccharide fucose into N-linked glycoproteins, a process known as "core fucosylation." Here we describe three unrelated individuals, who presented with intrauterine growth retardation, severe developmental and growth delays with shortened limbs, neurological impairments, and respiratory complications. Each underwent whole-exome sequencing and was found to carry pathogenic variants in FUT8. The first individual (consanguineous family) was homozygous for c.715C>T (p.Arg239∗), while the second (non-consanguineous family) was compound heterozygous for c.1009C>G (p.Arg337Gly) and a splice site variant c.1259+5G>T. The third individual (consanguineous family) was homozygous for a c.943C>T (p.Arg315∗). Splicing analysis confirmed the c.1259+5G>T resulted in expression of an abnormal FUT8 transcript lacking exon 9. Functional studies using primary fibroblasts from two affected individuals revealed a complete lack of FUT8 protein expression that ultimately resulted in substantial deficiencies in total core fucosylated N-glycans. Furthermore, serum samples from all three individuals showed a complete loss of core fucosylation. Here, we show that loss of function mutations in FUT8 cause a congenital disorder of glycosylation (FUT8-CDG) characterized by defective core fucosylation that phenotypically parallels some aspects of the Fut8-/- knockout mouse. Importantly, identification of additional affected individuals can be easily achieved through analysis of core fucosylation of N-glycans.

Using trio whole-exome sequencing, we have identified de novo heterozygous pathogenic variants in GRIA4 in five unrelated individuals with intellectual disability and other symptoms. GRIA4 encodes an AMPA receptor subunit known as GluR4, which is found on excitatory glutamatergic synapses and is important for learning and memory. Four of the variants are located in the highly conserved SYTANLAAF motif in the transmembrane protein M3, and the fifth is in an extra-cellular domain. Molecular modeling of the altered protein showed that three of the variants in the SYTANLAAF motif orient toward the center of the pore region and most likely lead to disturbance of the gating mechanism. The fourth variant in the SYTANLAAF motif most likely results in reduced permeability. The variant in the extracellular domain potentially interferes with the binding between the monomers. On the basis of clinical information and genetic results, and the fact that other subunits of the AMPA receptor have already been associated with neurodevelopmental disorders, we suggest that pathogenic de novo variants in GRIA4 lead to intellectual disability with or without seizures, gait abnormalities, problems of social behavior, and other variable features.

Meiotic recombination is essential to sexual reproduction and the generation of genetic diversity. Variation in recombination rates is presently of particular interest due to efforts being made to increase the rate of genetic gain in agricultural crops by breaking up large linkage disequilibrium blocks containing both beneficial and detrimental alleles. Here, a high-density genetic linkage map of Setaria was constructed using tunable genotyping by sequencing (tGBS) analysis of a population of recombinant inbred lines (RILs). Several regions of the Setaria genome exhibited significant levels of segregation distortion (SD), and recombination crossovers (COs) were also detected. The regions with high SD generally tended to have fewer COs, particularly for pericentromeric chromosomal areas. Recombination crossovers detected in Setaria were unevenly distributed across the genome and occurred more often in intergenic regions. Quantitative trait loci (QTLs) contributing towards the recombination frequency (Type I) and occurrence of COs in designated loci (Type II) were identified, and Type II QTLs garnered higher statistical power. The result of this study suggest that QTLs analysis of Type II traits using RILs might provide an opportunity to further understand meiotic recombination using high throughput genome sequencing and genotyping technologies.

With the expanded availability of next generation sequencing (NGS)-based clinical genetic tests, clinicians seeking to test patients with Mendelian diseases must weigh the superior coverage of targeted gene panels with the greater number of genes included in whole exome sequencing (WES) when considering their first-tier testing approach. Here, we use an in silico analysis to predict the analytic sensitivity of WES using pathogenic variants identified on targeted NGS panels as a reference.

Although genetic testing is increasingly used in clinical nephrology, a large number of patients with congenital abnormalities of the kidney and urinary tract (CAKUT) remain undiagnosed with current gene panels. Therefore, careful curation of novel genetic findings is key to improving diagnostic yields. We recently described a novel intellectual disability syndrome caused by de novo heterozygous loss-of-function mutations in the gene encoding the splicing factor SON. Here, we show that many of these patients, including two previously unreported, exhibit a wide array of kidney abnormalities. Detailed phenotyping of 14 patients with SON haploinsufficiency identified kidney anomalies in 8 patients, including horseshoe kidney, unilateral renal hypoplasia, and renal cysts. Recurrent urinary tract infections, electrolyte disturbances, and hypertension were also observed in some patients. SON knockdown in kidney cell lines leads to abnormal pre-mRNA splicing, resulting in decreased expression of several established CAKUT genes. Furthermore, these molecular events were observed in patient-derived cells with SON haploinsufficiency. Taken together, our data suggest that the wide spectrum of phenotypes in patients with a pathogenic SON mutation is a consequence of impaired pre-mRNA splicing of several CAKUT genes. We propose that genetic testing panels designed to diagnose children with a kidney phenotype should include the SON gene.

We clinically and molecularly characterize a new family with autosomal dominant rimmed vacuolar myopathy (RVM) caused by mutations in the HSPB8 gene.

Mutations in the short-chain enoyl-CoA hydratase (SCEH) gene, ECHS1, cause a rare autosomal recessive disorder of valine catabolism. Patients usually present with developmental delay, regression, dystonia, feeding difficulties, and abnormal MRI with bilateral basal ganglia involvement. We present clinical, biochemical, molecular, and functional data for four affected patients from two unrelated families of Samoan descent with identical novel compound heterozygous mutations. Family 1 has three affected boys while Family 2 has an affected daughter, all with clinical and MRI findings of Leigh syndrome and intermittent episodes of acidosis and ketosis. WES identified a single heterozygous variant in ECHS1 at position c.832G > A (p.Ala278Thr). However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT-PCR, RNA-Seq, RNA-Array) level identified a silent, common variant at position c.489G > A (p.Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.

Neutrophils are a type of white blood cell essential for the function of the innate immune system. To elucidate mechanisms of neutrophil biology, many studies are performed in vertebrate animal model systems. In Danio rerio (zebrafish), in vivo imaging of neutrophils is possible due to transgenic strains that possess fluorescently labeled leukocytes. However, due to the relative abundance of neutrophils, the counting process is laborious and subjective. In this article, we propose the use of a custom trained "you only look once" (YOLO) machine learning algorithm to automate the identification and counting of fluorescently labeled neutrophils in zebrafish. Using this model, we found the correlation coefficient between human counting and the model equals r = 0.8207 with an 8.65% percent error, while variation among human counters was 5% to 12%. Importantly, the model was able to correctly validate results of a previously published article that quantitated neutrophils manually. While the accuracy can be further improved, this model notably achieves these results in mere minutes compared with hours via standard manual counting protocols and can be performed by anyone with basic programming knowledge. It further supports the use of deep learning models for high throughput analysis of fluorescently labeled blood cells in the zebrafish model system.