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Genomic duplications in the SOX9 region are associated with human disease phenotypes; a study using human cells and mouse models reveals that the duplications can cause the formation of new higher-order chromatin structures called topologically associated domains (TADs) thereby resulting in changes in gene expression. Gene duplication and chromatin organization SOX9 is a developmental transcription factor with functions in chondrocyte differentiation and male sex determination, and genomic duplications in the SOX9 locus have been linked to various human diseases. Stefan Mundlos and colleagues use chromosome conformation capture techniques to look at the effect of such duplications on the chromatin partitioning units termed topologically associated domains (TADs) that surround the mouse Sox9 locus. They find that although TADs are stable genomic regulatory units, they can be rearranged by structural genomic variations to create novel chromatin regulatory domains. Duplications are generally thought to confer their phenotypic effect through an increase in gene dosage, but these results show how duplications can also affect higher order chromatin structure. Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions 1 , 2 . By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes 3 , 4 , 5 . However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans 6 , showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2 , in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

Corticotropin-independent Cushing's syndrome occurs with adrenocortical tumors or hyperplasia. The authors report that germline duplications of PRKACA lead to bilateral adrenal hyperplasia, whereas somatic mutations lead to unilateral cortisol-producing adrenal adenomas. Endogenous hypercortisolism, referred to as Cushing's syndrome, is associated with substantial morbidity and mortality. 1 When Cushing's syndrome is severe, patients have catabolic symptoms such as muscle weakness, skin fragility, osteoporosis, and severe metabolic sequelae. 2 Hypersecretion of cortisol can be driven by an excess of pituitary or ectopic corticotropin or can be due to adrenocortical tumors or hyperplasias with corticotropin-independent cortisol production. Adrenal adenomas are common, with a prevalence of at least 3% among persons older than 50 years of age. 3 Whereas only a subset of these tumors is associated with overt Cushing's syndrome, some degree of cortisol excess is present, . . .

The authors report that a gain of function in the catalytic subunit beta of the cyclic AMP–dependent protein kinase (protein kinase A), resulting from the presence of four copies of PRKACB (instead of the normal two), may lead to a Carney complex phenotype. To the Editor: Beuschlein et al. report a gain of function in PRKACA, the catalytic subunit alpha (Cα) of the cyclic AMP (cAMP)–dependent protein kinase (protein kinase A [PKA]) in cortisol-producing adenomas and micronodular adrenocortical hyperplasia. 1 Micronodular adrenocortical hyperplasia is also associated with Carney complex and inactivating mutations of the PKA regulatory subunit RIα (encoded by PRKAR1A ). 2 , 3 We report the case of a young woman with Carney complex who presented at 19 years of age with acromegaly, pigmented spots, and myxomas (Figure 1A); she did not have Cushing's syndrome (see the Supplementary Appendix, available with the full text of . . .

Split-Hand/Foot Malformation type 3 (SHFM3) is a congenital limb malformation associated with tandem duplications at the LBX1 / FGF8 locus. Yet, the disease patho-mechanism remains unsolved. Here we investigate the functional consequences of SHFM3-associated rearrangements on chromatin conformation and gene expression in vivo in transgenic mice. We show that the Lbx1 / Fgf8 locus consists of two separate, but interacting, regulatory domains. Re-engineering of a SHFM3-associated duplication and a newly reported inversion in mice results in restructuring of the chromatin architecture. This leads to ectopic activation of the Lbx1 and Btrc genes in the apical ectodermal ridge (AER) in an Fgf8- like pattern induced by AER-specific enhancers of Fgf8 . We provide evidence that the SHFM3 phenotype is the result of a combinatorial effect on gene misexpression in the developing limb. Our results reveal insights into the molecular mechanism underlying SHFM3 and provide conceptual framework for how genomic rearrangements can cause gene misexpression and disease. Congenital limb defects are often associated with genomic rearrangements. Here they provide insights into the molecular mechanism underlying SHFM3-associated structural variations, offering a conceptual framework for how genomic rearrangements can alter gene expression and cause disease.

Structural variants are a common cause of disease and contribute to a large extent to inter-individual variability, but their detection and interpretation remain a challenge. Here, we investigate 11 individuals with complex genomic rearrangements including germline chromothripsis by combining short- and long-read genome sequencing (GS) with Hi-C. Large-scale genomic rearrangements are identified in Hi-C interaction maps, allowing for an independent assessment of breakpoint calls derived from the GS methods, resulting in >300 genomic junctions. Based on a comprehensive breakpoint detection and Hi-C, we achieve a reconstruction of whole rearranged chromosomes. Integrating information on the three-dimensional organization of chromatin, we observe that breakpoints occur more frequently than expected in lamina-associated domains (LADs) and that a majority reshuffle topologically associating domains (TADs). By applying phased RNA-seq, we observe an enrichment of genes showing allelic imbalanced expression (AIG) within 100 kb around the breakpoints. Interestingly, the AIGs hit by a breakpoint (19/22) display both up- and downregulation, thereby suggesting different mechanisms at play, such as gene disruption and rearrangements of regulatory information. However, the majority of interpretable genes located 200 kb around a breakpoint do not show significant expression changes. Thus, there is an overall robustness in the genome towards large-scale chromosome rearrangements. Here the authors characterize structural variations (SVs) in a cohort of individuals with complex genomic rearrangements, identifying breakpoints by employing short- and long-read genome sequencing and investigate their impact on gene expression and the three-dimensional chromatin architecture. They find breakpoints are enriched in inactive regions and can result in chromatin domain fusions.

TDP2 encodes a 5′-tyrosyl DNA phosphodiesterase required for the efficient repair of double-strand breaks (DSBs) induced by the abortive activity of DNA topoisomerase II (TOP2). To date, only three homozygous variants in TDP2 have been reported in six patients from four unrelated pedigrees with spinocerebellar ataxia 23 (SCAR23). By whole-exome sequencing, we identified a novel TDP2 splice-site variant (c.636 + 3_636 + 6del) in two Italian siblings (aged 40 and 45) showing progressive ataxia, intellectual disability, speech delay, refractory seizures, and various physical anomalies. The variant caused exon 5 skipping with consequent nonsense-mediated mRNA decay and defective repair of TOP2-induced DSBs, as demonstrated by the functional assays on the patients’ fibroblasts. Our findings further demonstrate the pathogenic role of TDP2 biallelic loss-of-function variants in SCAR23 pathogenesis. Considering the age of our patients, the oldest reported to date, and their extensive follow-up, our study delineates in more detail the clinical phenotype related to the loss of TDP2 activity.

Meier-Gorlin syndrome (MGORS) is a rare disorder characterized by primordial dwarfism, microtia, and patellar aplasia/hypoplasia. Recessive mutations in ORC1, ORC4, ORC6, CDT1, CDC6, and CDC45, encoding members of the pre-replication (pre-RC) and pre-initiation (pre-IC) complexes, and heterozygous mutations in GMNN, a regulator of cell-cycle progression and DNA replication, have already been associated with this condition. We performed whole-exome sequencing (WES) in a patient with a clinical diagnosis of MGORS and identified biallelic variants in MCM5. This gene encodes a subunit of the replicative helicase complex, which represents a component of the pre-RC. Both variants, a missense substitution within a conserved domain critical for the helicase activity, and a single base deletion causing a frameshift and a premature stop codon, were predicted to be detrimental for the MCM5 function. Although variants of MCM5 have never been reported in specific human diseases, defect of this gene in zebrafish causes a phenotype of growth restriction overlapping the one associated with orc1 depletion. Complementation experiments in yeast showed that the plasmid carrying the missense variant was unable to rescue the lethal phenotype caused by mcm5 deletion. Moreover cell-cycle progression was delayed in patient's cells, as already shown for mutations in the ORC1 gene. Altogether our findings support the role of MCM5 as a novel gene involved in MGORS, further emphasizing that this condition is caused by impaired DNA replication.

Nasu-Hakola disease (NHD) is a rare autosomal recessive disorder characterized by progressive presenile dementia and bone cysts, caused by variants in either or . Despite the well-researched role of TREM2 and TYROBP/DAP12 in immunity, immunological phenotypes have never been reported in NHD patients. We initially diagnosed an Italian patient, using whole exome sequencing, with classical NHD clinical sequelae who additionally showed a decrease in NK cells and autoimmunity features underlined by the presence of autoantibodies. Based on this finding, we retrospectively explored the immunophenotype in another two NHD patients, in whom a low NK cell count and positive autoantibody serology were recorded. Accordingly, mice show abnormal levels of circulating proinflammatory cytokines and the dysfunction of immune cells, whereas knockout mice for , encoding the adapter for TREM2, exhibit increased levels of autoantibodies and defective NK cell activity. Our findings tend to redefine NHD as a multisystem \"immunological\" disease, considering that osteoclasts are derived from the fusion of mononuclear myeloid precursors, whereas neurological anomalies in NHD are directly caused by microglia dysfunction.

Most members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) families transduce signals via a canonical pathway involving the MyD88 adapter and the interleukin-1 receptor-associated kinase (IRAK) complex. This complex contains four molecules, including at least two (IRAK-1 and IRAK-4) active kinases. In mice and humans, deficiencies of IRAK-4 or MyD88 abolish most TLR (except for TLR3 and some TLR4) and IL-1R signaling in both leukocytes and fibroblasts. TLR and IL-1R responses are weak but not abolished in mice lacking IRAK-1, whereas the role of IRAK-1 in humans remains unclear. We describe here a boy with X-linked MECP2 deficiency-related syndrome due to a large de novo Xq28 chromosomal deletion encompassing both MECP2 and IRAK1. Like many boys with MECP2 null mutations, this child died very early, at the age of 7 mo. Unlike most IRAK-4– or MyD88-deficient patients, he did not suffer from invasive bacterial diseases during his short life. The IRAK-1 protein was completely absent from the patient’s fibroblasts, which responded very poorly to all TLR2/6 (PAM₂CSK₄, LTA, FSL-1), TLR1/2 (PAM₃CSK₄), and TLR4 (LPS, MPLA) agonists tested but had almost unimpaired responses to IL-1β. By contrast, the patient’s peripheral blood mononuclear cells responded normally to all TLR1/2, TLR2/6, TLR4, TLR7, and TLR8 (R848) agonists tested, and to IL-1β. The death of this child precluded long-term evaluations of the clinical consequences of inherited IRAK-1 deficiency. However, these findings suggest that human IRAK-1 is essential downstream from TLRs but not IL-1Rs in fibroblasts, whereas it plays a redundant role downstream from both TLRs and IL-1Rs in leukocytes.