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Familial hypertelorism, characterized by widely spaced eyes, classically shows autosomal dominant inheritance (Teebi type), but some pedigrees are compatible with X-linkage. No mechanism has been described previously, but clinical similarity has been noted to craniofrontonasal syndrome (CFNS), which is caused by mutations in the X-linked EFNB1 gene. Here we report a family in which females in three generations presented with hypertelorism, but lacked either craniosynostosis or a grooved nasal tip, excluding CFNS. DNA sequencing of EFNB1 was normal, but further analysis revealed a duplication of 937\u2009kb including EFNB1 and two flanking genes: PJA1 and STARD8. We found that the X chromosome bearing the duplication produces \u223c1.6-fold more EFNB1 transcript than the normal X chromosome and propose that, in the context of X-inactivation, this difference in expression level of EFNB1 results in abnormal cell sorting leading to hypertelorism. To support this hypothesis, we provide evidence from a mouse model carrying a targeted human EFNB1 cDNA, that abnormal cell sorting occurs in the cranial region. Hence, we propose that X-linked cases resembling Teebi hypertelorism may have a similar mechanism to CFNS, and that cellular mosaicism for different levels of ephrin-B1 (as well as simple presence/absence) leads to craniofacial abnormalities.
\n \n\n \n \nMuenke syndrome, defined by heterozygosity for a Pro250Arg substitution in fibroblast growth factor receptor 3 (FGFR3), is the most common genetic cause of craniosynostosis in humans. We have used gene targeting to introduce the Muenke syndrome mutation (equivalent to P244R) into the murine Fgfr3 gene. A rounded skull and shortened snout (often skewed) with dental malocclusion was observed in a minority of heterozygotes and many homozygotes. Development of this incompletely penetrant skull phenotype was dependent on genetic background and sex, with males more often affected. However, these cranial abnormalities were rarely attributable to craniosynostosis, which was only present in 2/364 mutants; more commonly, we found fusion of the premaxillary and/or zygomatic sutures. We also found decreased cortical thickness and bone mineral densities in long bones. We conclude that although both cranial and long bone development is variably affected by the murine Fgfr3(P244R) mutation, coronal craniosynostosis is not reliably reproduced.
\n \n\n \n \nCraniofrontonasal syndrome (CFNS), an X-linked disorder caused by loss-of-function mutations of EFNB1, exhibits a paradoxical sex reversal in phenotypic severity: females characteristically have frontonasal dysplasia, craniosynostosis and additional minor malformations, but males are usually more mildly affected with hypertelorism as the only feature. X-inactivation is proposed to explain the more severe outcome in heterozygous females, as this leads to functional mosaicism for cells with differing expression of EPHRIN-B1, generating abnormal tissue boundaries-a process that cannot occur in hemizygous males. Apparently challenging this model, males occasionally present with a more severe female-like CFNS phenotype. We hypothesized that such individuals might be mosaic for EFNB1 mutations and investigated this possibility in multiple tissue samples from six sporadically presenting males. Using denaturing high performance liquid chromatography, massively parallel sequencing and multiplex-ligation-dependent probe amplification (MLPA) to increase sensitivity above standard dideoxy sequencing, we identified mosaic mutations of EFNB1 in all cases, comprising three missense changes, two gene deletions and a novel point mutation within the 5' untranslated region (UTR). Quantification by Pyrosequencing and MLPA demonstrated levels of mutant cells between 15 and 69%. The 5' UTR variant mutates the stop codon of a small upstream open reading frame that, using a dual-luciferase reporter construct, was demonstrated to exacerbate interference with translation of the wild-type protein. These results demonstrate a more severe outcome in mosaic than in constitutionally deficient males in an X-linked dominant disorder and provide further support for the cellular interference mechanism, normally related to X-inactivation in females.
\n \n\n \n \nCraniofrontonasal syndrome (CFNS) is an X-linked developmental disorder that shows paradoxically greater severity in heterozygous females than in hemizygous males. Females have frontonasal dysplasia and coronal craniosynostosis (fusion of the coronal sutures); in males, hypertelorism is the only typical manifestation. Here, we show that the classical female CFNS phenotype is caused by heterozygous loss-of-function mutations in EFNB1, which encodes a member of the ephrin family of transmembrane ligands for Eph receptor tyrosine kinases. In mice, the orthologous Efnb1 gene is expressed in the frontonasal neural crest and demarcates the position of the future coronal suture. Although EFNB1 is X-inactivated, we did not observe markedly skewed X-inactivation in either blood or cranial periosteum from females with CFNS, indicating that lack of ephrin-B1 does not compromise cell viability in these tissues. We propose that in heterozygous females, patchwork loss of ephrin-B1 disturbs tissue boundary formation at the developing coronal suture, whereas in males deficient in ephrin-B1, an alternative mechanism maintains the normal boundary. This is the only known mutation in the ephrin/Eph receptor signaling system in humans and provides clues to the biogenesis of craniosynostosis.
\n \n\n \n \nThe mouse mutant Doublefoot (Dbf) shows preaxial polydactyly with 6-9 triphalangeal digits in all four limbs and additional abnormalities including a broadened skull, hydrocephalus, and a thickened, kinked tail. The autopod undergoes a characteristic expansion between late embryonic day (E) 10.5 and E11.5, following the onset of ectopic Indian hedgehog (Ihh) expression in the entire distal mesenchyme, except for the zone of polarising activity (ZPA), at E10.5. We show here that limb prepattern, as indicated by expression of Gli3 and Hand2 at E9.5 is unaffected by the mutation. As both Sonic hedgehog (Shh) and Ihh expression are present in Dbf limb buds at E10.5, we generated Dbf/(+);Shh(-/-) mutants to analyse the effects of different patterns of Hedgehog activity on the limb phenotype and molecular differentiation. Dbf/(+) embryos lacking Shh showed postaxial as well as preaxial polydactyly, and the Ihh expression domain extended posteriorly into the domain in which Shh is normally expressed, indicating loss of ZPA identity. Differences in gene expression patterns in wild type, single and compound mutants were associated with differences in Gli3 processing: an increased ratio of Gli3 activator to Gli3 repressor was observed in the anterior half of Dbf/(+) limb buds and in both anterior and posterior halves of compound mutant limb buds at E10.5. To identify the cause of Ihh misregulation in Dbf/(+) mutants, we sequenced approximately 20 kb of genomic DNA around Ihh but found no pathogenic changes. However, Southern blot analysis revealed a approximately 600 kb deletion disrupting or deleting 25 transcripts, starting 50 kb 5' of Ihh and extending away from the gene. The large deletion interval may explain the wide range of abnormalities in Dbf/(+) mutants. However, we did not detect anologous deletions in cases of Laurin-Sandrow syndrome, a human disorder that shows phenotypic similarities to Dbf.
\n \n\n \n \nAccurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next-generation-sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical-grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically-reliable diagnostic test and minimize false-negative results we developed an open-source tool (CoverMi) to accurately determine base-coverage and the 'discoverability' of known mutations for every sample. We validated our 33-gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99\u00b77% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38\u00b76%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS-based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.
\n \n\n \n \n\n Intrathecal synthesis of central nervous system (CNS)-reactive autoantibodies is observed across patients with autoimmune encephalitis (AE), who show multiple residual neurobehavioral deficits and relapses despite immunotherapies. We leveraged two common forms of AE, mediated by leucine-rich glioma inactivated-1 (LGI1) and contactin-associated protein-like 2 (CASPR2) antibodies, as human models to comprehensively reconstruct and profile cerebrospinal fluid (CSF) B cell receptor (BCR) characteristics. We hypothesized that the resultant observations would both inform the observed therapeutic gap and determine the contribution of intrathecal maturation to pathogenic B cell lineages. From the CSF of three patients, 381 cognate-paired IgG BCRs were isolated by cell sorting and scRNA-seq, and 166 expressed as monoclonal antibodies (mAbs). Sixty-two percent of mAbs from singleton BCRs reacted with either LGI1 or CASPR2 and, strikingly, this rose to 100% of cells in clonal groups with \u22654 members. These autoantigen-reactivities were more concentrated within antibody-secreting cells (ASCs) versus B cells (\n P\n < 0.0001), and both these cell types were more differentiated than LGI1- and CASPR2-unreactive counterparts. Despite greater differentiation, autoantigen-reactive cells had acquired few mutations intrathecally and showed minimal variation in autoantigen affinities within clonal expansions. Also, limited CSF T cell receptor clonality was observed. In contrast, a comparison of germline-encoded BCRs versus the founder intrathecal clone revealed marked gains in both affinity and mutational distances (\n P\n = 0.004 and\n P\n < 0.0001, respectively). Taken together, in patients with LGI1 and CASPR2 antibody encephalitis, our results identify CSF as a compartment with a remarkably high frequency of clonally expanded autoantigen-reactive ASCs whose BCR maturity appears dominantly acquired outside the CNS.\n
\n \n\n \n \nWhilst the regulation of chromatin accessibility and its effect on gene expression have been well studied in eukaryotic species, the role of chromatin dynamics and 3D organisation in genome reduced bacteria remains poorly understood [1,2]. In this study we profiled the accessibility of the Mycoplasma hyorhinis genome, these data were collected fortuitously as part of an experiment where ATAC-Seq was conducted on mycoplasma, contaminated mammalian cells. We found a differential and highly reproducible chromatin accessibility landscape, with regions of increased accessibility corresponding to genes important for the bacteria's life cycle and infectivity. Furthermore, accessibility in general correlated with transcriptionally active genes as profiled by RNA-Seq, but peaks of high accessibility were also seen in non-coding and intergenic regions, which could contribute to the topological organisation of the genome. However, changes in transcription induced by starvation or application of the RNA polymerase inhibitor rifampicin did not themselves change the accessibility profile, which confirms that the differential accessibility is inherently a property of the genome, and not a consequence of its function. These results together show that differential chromatin accessibility is a key feature of the regulation of gene expression in bacteria.
\n \n\n \n \nBACKGROUND: SMAD6 encodes an intracellular inhibitor of the bone morphogenetic protein (BMP) signalling pathway. Until now, rare heterozygous loss-of-function variants in SMAD6 were demonstrated to increase the risk of disparate clinical disorders including cardiovascular disease, craniosynostosis and radioulnar synostosis. Only two unrelated patients harbouring biallelic SMAD6 variants presenting a complex cardiovascular phenotype and facial dysmorphism have been described. CASES: Here, we present the first two patients with craniosynostosis harbouring homozygous SMAD6 variants. The male probands, both born to healthy consanguineous parents, were diagnosed with metopic synostosis and bilateral or unilateral radioulnar synostosis. Additionally, one proband had global developmental delay. Echocardiographic evaluation did not reveal cardiac or outflow tract abnormalities. MOLECULAR ANALYSES: The novel missense (c.[584T>G];[584T>G], p.[(Val195Gly)];[(Val195Gly)]) and missense/splice-site variant (c.[817G>A];[817G>A], r.[(817g>a,817delins[a;817+2_817+228])];[(817g>a,817delins[a;817+2_817+228])], p.[(Glu273Lys,Glu273Serfs*72)];[(Glu273Lys,Glu273Serfs*72)]) both locate in the functional MH1 domain of the protein and have not been reported in gnomAD database. Functional analyses of the variants showed reduced inhibition of BMP signalling or abnormal splicing, respectively, consistent with a hypomorphic mechanism of action. CONCLUSION: Our data expand the spectrum of variants and phenotypic spectrum associated with homozygous variants of SMAD6 to include craniosynostosis.
\n \n\n \n \nBACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) has rapidly become the most common liver disease world-wide. Modern lifestyles have been linked to this rise in prevalence with changes in rhythmic human behaviour emerging as a possible mechanism. We investigated how shift working patterns and chronotype were associated with hepatic fat fraction and NAFLD in 282,303 UK Biobank participants. METHODS: We stratified participants into day, irregular-shift, and permanent night-shift workers. We then utilized multiple methods of disease identification including: a) Dallas Steatosis Index (DSI), b) ICD10 codes, and c) hepatic proton density fat fraction (PDFF) and examined how shiftwork exposure impacted these variables. We further assessed the relationship of baseline chronotype with liver phenotypes using these same outcome measures. RESULTS: Compared to day workers, irregular-shift workers were more likely to have a high DSI (OR 1.29 [1.2-1.4]) after adjusting for major covariates with some attenuation after additional adjustment for BMI (OR 1.12 [1.03-1.22]). Likelihood of high DSI was also increased in permanent night-shift workers (OR 1.08 [0.9-1.29]) in the fully-adjusted model. Mediator analysis revealed that BMI was a significant mediator of the shiftwork effect. Compared to participants with intermediate chronotype, those with extreme late chronotype had a higher likelihood of high DSI defined NAFLD (OR 1.45 [1.34 -1.56]) and a higher likelihood of NAFLD/NASH by ICD10 code (OR 1.23 [1.09-1.39]). Hepatic PDFF was elevated in irregular shift workers, but not permanent night shift workers. CONCLUSIONS: Irregular-shift work and extreme late chronotype are associated with pathological liver fat accumulation, suggesting circadian misalignment may have an underlying pathogenic role. These findings have implications for health interventions to mitigate the detrimental effect of shift work.
\n \n\n \n \nThe severe virulence of Toxoplasma gondii in classical laboratory inbred mouse strains contradicts the hypothesis that house mice (Mus musculus) are the most important intermediate hosts for its transmission and evolution because death of the mouse before parasite transmission equals death of the parasite. However, the classical laboratory inbred mouse strains (Mus musculus domesticus), commonly used to test Toxoplasma strain differences in virulence, do not capture the genetic diversity within Mus musculus. Thus, it is possible that Toxoplasma strains that are severely virulent in laboratory inbred mice are avirulent in some other mouse sub-species. Here, we present insight into the responses of individual mouse strains, representing strains of the genetically divergent Mus musculus musculus, Mus musculus castaneus and Mus musculus domesticus, to infection with individual clonal and atypical Toxoplasma strains. We observed that, unlike M. m. domesticus, M. m. musculus and M. m. castaneus are resistant to the clonal Toxoplasma strains. For M. m. musculus, we show that this is due to a locus on chromosome 11 that includes the genes that encode the interferon gamma (IFNG)-inducible immunity-related GTPases (Irgs) that can kill the parasite by localising and subsequently vesiculating the parasitophorous vacuole membrane. However, despite the localization of known effector Irgs to the Toxoplasma parasitophorous vacuole membrane, we observed that some atypical Toxoplasma strains are virulent in all the mouse strains tested. The virulence of these atypical strains in M. m. musculus could not be attributed to individual rhoptry protein 5 (ROP5) alleles, a secreted parasite pseudokinase that antagonises the canonical effector Irgs and is indispensable for parasite virulence in laboratory inbred mice (M. m. domesticus). We conclude that murine resistance to Toxoplasma is modulated by complex interactions between host and parasite genotypes and may be independent of known effector Irgs on murine chromosome 11.
\n \n\n \n \n<p>Supplementary Table 6 qRT PCR Probes</p>
\n \n\n \n \n<p>Supplementary Table 1 VEGFAC Top Differentially Expressed Genes by Cluster</p>
\n \n\n \n \n<p>Supplementary Materials and Methods, Supplementary Figures S1-S12</p>
\n \n\n \n \n<div>Abstract<p>A lack of models that recapitulate the complexity of human bone marrow has hampered mechanistic studies of normal and malignant hematopoiesis and the validation of novel therapies. Here, we describe a step-wise, directed-differentiation protocol in which organoids are generated from induced pluripotent stem cells committed to mesenchymal, endothelial, and hematopoietic lineages. These 3D structures capture key features of human bone marrow\u2014stroma, lumen-forming sinusoids, and myeloid cells including proplatelet-forming megakaryocytes. The organoids supported the engraftment and survival of cells from patients with blood malignancies, including cancer types notoriously difficult to maintain <i>ex vivo</i>. Fibrosis of the organoid occurred following TGF\u03b2 stimulation and engraftment with myelofibrosis but not healthy donor\u2013derived cells, validating this platform as a powerful tool for studies of malignant cells and their interactions within a human bone marrow\u2013like milieu. This enabling technology is likely to accelerate the discovery and prioritization of novel targets for bone marrow disorders and blood cancers.</p>Significance:<p>We present a human bone marrow organoid that supports the growth of primary cells from patients with myeloid and lymphoid blood cancers. This model allows for mechanistic studies of blood cancers in the context of their microenvironment and provides a much-needed <i>ex vivo</i> tool for the prioritization of new therapeutics.</p><p><i><a href=\"https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-22-1303\" target=\"_blank\">See related commentary by Derecka and Crispino, p. 263</a>.</i></p><p><i><a href=\"https://aacrjournals.org/cancerdiscovery/article/doi/10.1158/2159-8290.CD-13-2-ITI\" target=\"_blank\">This article is highlighted in the In This Issue feature, p. 247</a></i></p></div>
\n \n\n \n \n<p>Supplementary Table 2 Gene sets for GSEA</p>
\n \n\n \n \n<p>Supplementary Table 5 Antibodies</p>
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