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Germinal centers (GCs) form in lymph nodes after immunization or infection to facilitate antibody affinity maturation and memory and plasma cell (PC) development. PC differentiation is thought to involve stringent selection for GC B cells expressing the highest-affinity antigen receptors, but how this plays out during complex polyclonal responses is unclear. We combine temporal lineage tracing with antibody characterization to gain a snapshot of PCs developing during influenza infection. GCs co-mature B cell clones with antibody affinities spanning multiple orders of magnitude; however, each generates PCs with similar efficiencies, including weak binders. Within lineages, PC selection is not restricted to variants with the highest-affinity antibodies. Differentiation is commonly associated with proliferative expansion to produce "nodes" of identical PCs. Immunization-induced GCs generate fewer PCs but still of low- and high-antibody affinities. We propose that generating low-affinity antibody PCs reflects an evolutionary compromise to facilitate diverse serum antibody responses. [Display omitted] • Germinal center B cells are fate-mapped to study plasma cell selection during infection • Antibody affinities of PCs simultaneously exiting GCs span several orders of magnitude • Substantial GC clonal diversity is retained through PC selection • GC PC output after protein immunization is much reduced but remains affinity permissive B cells are known to undergo sequential rounds of mutation and affinity-based selection, in sites known as germinal centers, to produce higher-affinity antibodies over time. This study finds that germinal centers formed in response to complex immune challenges support the differentiation of clonally diverse plasma cells, including some of low-antibody affinity, suggesting that high affinity is not the only factor driving the maturation of the antibody repertoire. [ABSTRACT FROM AUTHOR]

DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G 1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1 , ORC4 , ORC6 , CDT1 , or CDC6 ) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN , cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis. [ABSTRACT FROM AUTHOR]

Curry-Jones syndrome (CJS) is a multisystem disorder characterized by patchy skin lesions, polysyndactyly, diverse cerebral malformations, unicoronal craniosynostosis, iris colobomas, microphthalmia, and intestinal malrotation with myofibromas or hamartomas. Cerebellar medulloblastoma has been described in a single affected individual; in another, biopsy of skin lesions showed features of trichoblastoma. The combination of asymmetric clinical features, patchy skin manifestations, and neoplastic association previously led to the suggestion that this could be a mosaic condition, possibly involving hedgehog (Hh) signaling. Here, we show that CJS is caused by recurrent somatic mosaicism for a nonsynonymous variant in SMO (c.1234C>T [p.Leu412Phe]), encoding smoothened (SMO), a G-protein-coupled receptor that transduces Hh signaling. We identified eight mutation-positive individuals (two of whom had not been reported previously) with highly similar phenotypes and demonstrated varying amounts of the mutant allele in different tissues. We present detailed findings from brain MRI in three mutation-positive individuals. Somatic SMO mutations that result in constitutive activation have been described in several tumors, including medulloblastoma, ameloblastoma, and basal cell carcinoma. Strikingly, the most common of these mutations is the identical nonsynonymous variant encoding p.Leu412Phe. Furthermore, this substitution has been shown to activate SMO in the absence of Hh signaling, providing an explanation for tumor development in CJS. This raises therapeutic possibilities for using recently generated Hh-pathway inhibitors. In summary, our work uncovers the major genetic cause of CJS and illustrates strategies for gene discovery in the context of low-level tissue-specific somatic mosaicism. [ABSTRACT FROM AUTHOR]

Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired , encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2 , in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5–12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1 , which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded. [ABSTRACT FROM AUTHOR]

Richieri-Costa-Pereira syndrome is an autosomal-recessive acrofacial dysostosis characterized by mandibular median cleft associated with other craniofacial anomalies and severe limb defects. Learning and language disabilities are also prevalent. We mapped the mutated gene to a 122 kb region at 17q25.3 through identity-by-descent analysis in 17 genealogies. Sequencing strategies identified an expansion of a region with several repeats of 18- or 20-nucleotide motifs in the 5′ untranslated region (5′ UTR) of EIF4A3, which contained from 14 to 16 repeats in the affected individuals and from 3 to 12 repeats in 520 healthy individuals. A missense substitution of a highly conserved residue likely to affect the interaction of eIF4AIII with the UPF3B subunit of the exon junction complex in trans with an expanded allele was found in an unrelated individual with an atypical presentation, thus expanding mutational mechanisms and phenotypic diversity of RCPS. EIF4A3 transcript abundance was reduced in both white blood cells and mesenchymal cells of RCPS-affected individuals as compared to controls. Notably, targeting the orthologous eif4a3 in zebrafish led to underdevelopment of several craniofacial cartilage and bone structures, in agreement with the craniofacial alterations seen in RCPS. Our data thus suggest that RCPS is caused by mutations in EIF4A3 and show that EIF4A3, a gene involved in RNA metabolism, plays a role in mandible, laryngeal, and limb morphogenesis. [ABSTRACT FROM AUTHOR]

Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed. [Copyright &y& Elsevier]

Congenital myasthenic syndromes are a heterogeneous group of inherited disorders that arise from impaired signal transmission at the neuromuscular synapse. They are characterized by fatigable muscle weakness. We performed whole-exome sequencing to determine the underlying defect in a group of individuals with an inherited limb-girdle pattern of myasthenic weakness. We identify DPAGT1 as a gene in which mutations cause a congenital myasthenic syndrome. We describe seven different mutations found in five individuals with DPAGT1 mutations. The affected individuals share a number of common clinical features, including involvement of proximal limb muscles, response to treatment with cholinesterase inhibitors and 3,4-diaminopyridine, and the presence of tubular aggregates in muscle biopsies. Analyses of motor endplates from two of the individuals demonstrate a severe reduction of endplate acetylcholine receptors. DPAGT1 is an essential enzyme catalyzing the first committed step of N-linked protein glycosylation. Our findings underscore the importance of N-linked protein glycosylation for proper functioning of the neuromuscular junction. Using the DPAGT1-specific inhibitor tunicamycin, we show that DPAGT1 is required for efficient glycosylation of acetylcholine-receptor subunits and for efficient export of acetylcholine receptors to the cell surface. We suggest that the primary pathogenic mechanism of DPAGT1 mutations is reduced levels of acetylcholine receptors at the endplate region. These individuals share clinical features similar to those of congenital myasthenic syndrome due to GFPT1 mutations, and their disorder might be part of a larger subgroup comprising the congenital myasthenic syndromes that result from defects in the N-linked glycosylation pathway and that manifest through impaired neuromuscular transmission. [Copyright &y& Elsevier]

Summary: A substantial amount of organismal complexity is thought to be encoded by enhancers which specify the location, timing, and levels of gene expression. In mammals there are more enhancers than promoters which are distributed both between and within genes. Here we show that activated, intragenic enhancers frequently act as alternative tissue-specific promoters producing a class of abundant, spliced, multiexonic poly(A)+ RNAs (meRNAs) which reflect the host gene''s structure. meRNAs make a substantial and unanticipated contribution to the complexity of the transcriptome, appearing as alternative isoforms of the host gene. The low protein-coding potential of meRNAs suggests that many meRNAs may be byproducts of enhancer activation or underlie as-yet-unidentified RNA-encoded functions. Distinguishing between meRNAs and mRNAs will transform our interpretation of dynamic changes in transcription both at the level of individual genes and of the genome as a whole. [Copyright &y& Elsevier]

Summary Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for resolving transcriptional heterogeneity. However, its application to studying cancerous tissues is currently hampered by the lack of coverage across key mutation hotspots in the vast majority of cells; this lack of coverage prevents the correlation of genetic and transcriptional readouts from the same single cell. To overcome this, we developed TARGET-seq, a method for the high-sensitivity detection of multiple mutations within single cells from both genomic and coding DNA, in parallel with unbiased whole-transcriptome analysis. Applying TARGET-seq to 4,559 single cells, we demonstrate how this technique uniquely resolves transcriptional and genetic tumor heterogeneity in myeloproliferative neoplasms (MPN) stem and progenitor cells, providing insights into deregulated pathways of mutant and non-mutant cells. TARGET-seq is a powerful tool for resolving the molecular signatures of genetically distinct subclones of cancer cells. Graphical Abstract Highlights • Conventional scRNA-seq protocols do not allow reliable mutational analysis • TARGET-seq combines high-sensitivity genomic DNA and cDNA genotyping with scRNA-seq • TARGET-seq resolves the distinct transcriptional signatures of tumor genetic subclones • Non-mutant cells from patients show aberrant, inflammation-associated gene expression Rodriguez-Meira et al. developed TARGET-seq, a method for high-sensitivity mutational analysis and parallel RNA sequencing from the same single cell. Applied to 4,559 single cells, TARGET-seq unraveled transcriptional and genetic tumor heterogeneity in myeloproliferative neoplasm (MPN) stem and progenitor cells. TARGET-seq is a powerful tool for resolving the molecular signatures of genetically distinct tumor subclones. [ABSTRACT FROM AUTHOR]

Adaptive immunity involves the development of bespoke antibodies in germinal centers (GCs) through immunoglobulin somatic hypermutation (SHM) in GC dark zones (DZs) and clonal selection in light zones (LZs). Accurate selection requires that cells fully replace surface B cell receptors (BCRs) following SHM, but whether this happens before LZ entry is not clear. We found that most GC B cells degrade pre-SHM receptors before leaving the DZ, and that B cells acquiring crippling mutations during SHM rarely reached the LZ. Instead, apoptosis was triggered preferentially in late G1, a stage wherein cells with functional BCRs re-entered cell cycle or reduced surface expression of the chemokine receptor CXCR4 to enable LZ migration. Ectopic expression of the anti-apoptotic gene Bcl2 was not sufficient for cells with damaging mutations to reach the LZ, suggesting that BCR-dependent cues may actively facilitate the transition. Thus, BCR replacement and pre-screening in DZs prevents the accumulation of clones with non-functional receptors and facilitates selection in the LZ., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)