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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.

The protein FAM134B is an endoplasmic reticulum (ER)-resident receptor that facilitates ER autophagy, and downregulation of this protein (mutations of which are also known to cause sensory neuropathy in humans) results in expanded ER structures and degeneration of mouse sensory neurons. ER-phagy receptors mapped The endoplasmic reticulum (ER) is a complex network of membranes involved in protein and lipid synthesis, ion homeostasis, protein quality control and organelle communication. It is also a source of membrane-bounded vesicles called autophagosomes, the vehicles for the self-digesting cellular process of autophagy. Two papers published online this week [in this issue of Nature] show how the ER itself is targeted for degradation by autophagy — a process that could ensure constant ER turnover in response to cellular requirements. Ivan Dikic and coworkers find the protein FAM134B is an ER-resident receptor that facilitates 'ER-phagy'. Downregulation of this protein — mutations of which can cause sensory neuropathy in humans — resulted in expanded ER structures and degeneration of mouse sensory neurons. Hitoshi Nakatogawa and colleagues show that the same phenomenon is conserved in yeast, where Atg40 is enriched in the cortical and cytoplasmic ER, loading these ER subdomains into autophagosomes. A further ER-phagy receptor, Atg39, localizes to the perinuclear ER (or the nuclear envelope) and induces autophagic sequestration of a part of the nucleus, thus ensuring cell survival under nitrogen-deprived conditions. The endoplasmic reticulum (ER) is the largest intracellular endomembrane system, enabling protein and lipid synthesis, ion homeostasis, quality control of newly synthesized proteins and organelle communication 1 . Constant ER turnover and modulation is needed to meet different cellular requirements and autophagy has an important role in this process 2 , 3 , 4 , 5 , 6 , 7 , 8 . However, its underlying regulatory mechanisms remain unexplained. Here we show that members of the FAM134 reticulon protein family are ER-resident receptors that bind to autophagy modifiers LC3 and GABARAP, and facilitate ER degradation by autophagy (‘ER-phagy’). Downregulation of FAM134B protein in human cells causes an expansion of the ER, while FAM134B overexpression results in ER fragmentation and lysosomal degradation. Mutant FAM134B proteins that cause sensory neuropathy in humans 9 are unable to act as ER-phagy receptors. Consistently, disruption of Fam134b in mice causes expansion of the ER, inhibits ER turnover, sensitizes cells to stress-induced apoptotic cell death and leads to degeneration of sensory neurons. Therefore, selective ER-phagy via FAM134 proteins is indispensable for mammalian cell homeostasis and controls ER morphology and turnover in mice and humans.

Understanding the dark genome is a priority task following the complete sequencing of the human genome. Short open reading frames (sORFs) are a group of largely unexplored elements of the dark genome with the potential for being translated into microproteins. The definitive number of coding and regulatory sORFs is not known, however they could account for up to 1–2% of the human genome. This corresponds to an order of magnitude in the range of canonical coding genes. For a few sORFs a clinical relevance has already been demonstrated, but for the majority of potential sORFs the biological function remains unclear. A major limitation in predicting their disease relevance using large-scale genomic data is the fact that no population-level constraint metrics for genetic variants in sORFs are yet available. To overcome this, we used the recently released gnomAD 4.0 dataset and analyzed the constraint of a consensus set of sORFs and their genomic neighbors. We demonstrate that sORFs are mostly embedded into a moderately constrained genomic context, but within the gencode dataset we identified a subset of highly constrained sORFs comparable to highly constrained canonical genes.

Familial Adult Myoclonic Epilepsy (FAME) is a genetically heterogeneous disorder characterized by cortical tremor and seizures. Intronic TTTTA/TTTCA repeat expansions in SAMD12 (FAME1) are the main cause of FAME in Asia. Using genome sequencing and repeat-primed PCR, we identify another site of this repeat expansion, in MARCH6 (FAME3) in four European families. Analysis of single DNA molecules with nanopore sequencing and molecular combing show that expansions range from 3.3 to 14 kb on average. However, we observe considerable variability in expansion length and structure, supporting the existence of multiple expansion configurations in blood cells and fibroblasts of the same individual. Moreover, the largest expansions are associated with micro-rearrangements occurring near the expansion in 20% of cells. This study provides further evidence that FAME is caused by intronic TTTTA/TTTCA expansions in distinct genes and reveals that expansions exhibit an unexpectedly high somatic instability that can ultimately result in genomic rearrangements. Familial cortical myoclonic tremor with epilepsy (FAME) is a slowly progressing cortical tremor mapping to various genomic loci, including intronic expansions in SAMD12 for FAME1. Here, Florian et al. describe mixed intronic TTTTA/TTTCA expansions of various lengths in the first intron of MARCH6 as a cause of FAME3.

Background Methods to call, analyze and visualize copy number variations (CNVs) from massive parallel sequencing data have been widely adopted in clinical practice and genetic research. To enable a streamlined analysis of CNV data, comprehensive annotations and good visualizations are indispensable. The ability to detect single exon CNVs is another important feature for genetic testing. Nonetheless, most available open-source tools come with limitations in at least one of these areas. One additional drawback is that available tools deliver data in an unstructured and static format which requires subsequent visualization and formatting efforts. Results Here we present CNVizard, an interactive Streamlit app allowing a comprehensive visualization of CNVkit data. Furthermore, combining CNVizard with the CNVand pipeline allows the annotation and visualization of CNV or SV VCF files from any CNV caller. Conclusion CNVizard, in combination with CNVand, enables the comprehensive and streamlined analysis of short- and long-read sequencing data and provide an intuitive webapp-like experience enabling an interactive visualization of CNV data.

Background and aims: Small fiber neuropathy (SFN) is increasingly suspected in patients with pain of uncertain origin, and making the diagnosis remains a challenge lacking a diagnostic gold standard. Methods: In this case–control study, we prospectively recruited 86 patients with a medical history and clinical phenotype suggestive of SFN. Patients underwent neurological examination, quantitative sensory testing (QST), and distal and proximal skin punch biopsy, and were tested for pain-associated gene loci. Fifty-five of these patients additionally underwent pain-related evoked potentials (PREP), corneal confocal microscopy (CCM), and a quantitative sudomotor axon reflex test (QSART). Results: Abnormal distal intraepidermal nerve fiber density (IENFD) (60/86, 70%) and neurological examination (53/86, 62%) most frequently reflected small fiber disease. Adding CCM and/or PREP further increased the number of patients with small fiber impairment to 47/55 (85%). Genetic testing revealed potentially pathogenic gene variants in 14/86 (16%) index patients. QST, QSART, and proximal IENFD were of lower impact. Conclusion: We propose to diagnose SFN primarily based on the results of neurological examination and distal IENFD, with more detailed phenotyping in specialized centers.

Adult kidney organoids have been described as strictly tubular epithelia and termed tubuloids. While the cellular origin of tubuloids has remained elusive, here we report that they originate from a distinct CD24 + epithelial subpopulation. Long-term-cultured CD24 + cell-derived tubuloids represent a functional human kidney tubule. We show that kidney tubuloids can be used to model the most common inherited kidney disease, namely autosomal dominant polycystic kidney disease (ADPKD), reconstituting the phenotypic hallmark of this disease with cyst formation. Single-cell RNA sequencing of CRISPR–Cas9 gene-edited PKD1- and PKD2 -knockout tubuloids and human ADPKD and control tissue shows similarities in upregulation of disease-driving genes. Furthermore, in a proof of concept, we demonstrate that tolvaptan, the only approved drug for ADPKD, has a significant effect on cyst size in tubuloids but no effect on a pluripotent stem cell-derived model. Thus, tubuloids are derived from a tubular epithelial subpopulation and represent an advanced system for ADPKD disease modeling. Adult kidney organoids, or tubuloids, originate from CD24 + epithelial cells. Tubuloids represent a functional kidney tubule and can be used to model autosomal dominant polycystic kidney disease and study drug response.

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by an expanded polyglutamine repeat in the huntingtin gene. The neuropathology of HD is characterized by the decline of a specific neuronal population within the brain, the striatal medium spiny neurons (MSNs). The origins of this extreme vulnerability remain unknown. Human induced pluripotent stem cell (hiPS cell)-derived MSNs represent a powerful tool to study this genetic disease. However, the differentiation protocols published so far show a high heterogeneity of neuronal populations in vitro. Here, we compared two previously published protocols to obtain hiPS cell-derived striatal neurons from both healthy donors and HD patients. Patch-clamp experiments, immunostaining and RT-qPCR were performed to characterize the neurons in culture. While the neurons were mature enough to fire action potentials, a majority failed to express markers typical for MSNs. Voltage-clamp experiments on voltage-gated sodium (Nav) channels revealed a large variability between the two differentiation protocols. Action potential analysis did not reveal changes induced by the HD mutation. This study attempts to demonstrate the current challenges in reproducing data of previously published differentiation protocols and in generating hiPS cell-derived striatal MSNs to model a genetic neurodegenerative disorder in vitro.

Sphingolipids are important components of cellular membranes and functionally associated with fundamental processes such as cell differentiation, neuronal signaling, and myelin sheath formation. Defects in the synthesis or degradation of sphingolipids leads to various neurological pathologies; however, the entire spectrum of sphingolipid metabolism disorders remains elusive. A combined approach of genomics and lipidomics was applied to identify and characterize a human sphingolipid metabolism disorder. By whole-exome sequencing in a patient with a multisystem neurological disorder of both the central and peripheral nervous systems, we identified a homozygous p.Ala280Val variant in DEGS1, which catalyzes the last step in the ceramide synthesis pathway. The blood sphingolipid profile in the patient showed a significant increase in dihydro sphingolipid species that was further recapitulated in patient-derived fibroblasts, in CRISPR/Cas9-derived DEGS1-knockout cells, and by pharmacological inhibition of DEGS1. The enzymatic activity in patient fibroblasts was reduced by 80% compared with wild-type cells, which was in line with a reduced expression of mutant DEGS1 protein. Moreover, an atypical and potentially neurotoxic sphingosine isomer was identified in patient plasma and in cells expressing mutant DEGS1. We report DEGS1 dysfunction as the cause of a sphingolipid disorder with hypomyelination and degeneration of both the central and peripheral nervous systems. Not applicable. Seventh Framework Program of the European Commission, Swiss National Foundation, Rare Disease Initiative Zurich.

Ingo Kurth and colleagues show that a specific de novo missense mutation in SCN11A results in an inability to experience pain. They further show that mutant channels display higher activity at resting voltages, causing sustained depolarization of pain receptors, impaired generation of action potentials and aberrant synaptic transmission. The sensation of pain protects the body from serious injury 1 , 2 , 3 . Using exome sequencing, we identified a specific de novo missense mutation in SCN11A in individuals with the congenital inability to experience pain who suffer from recurrent tissue damage and severe mutilations. Heterozygous knock-in mice carrying the orthologous mutation showed reduced sensitivity to pain and self-inflicted tissue lesions, recapitulating aspects of the human phenotype. SCN11A encodes Na v 1.9, a voltage-gated sodium ion channel that is primarily expressed in nociceptors, which function as key relay stations for the electrical transmission of pain signals from the periphery to the central nervous system 4 , 5 . Mutant Na v 1.9 channels displayed excessive activity at resting voltages, causing sustained depolarization of nociceptors, impaired generation of action potentials and aberrant synaptic transmission. The gain-of-function mechanism that underlies this channelopathy suggests an alternative way to modulate pain perception.