2022

British Journal of Dermatology

A homozygous p.Leu813Pro gain-of-function NLRP1 variant causes phenotypes of different severity in two siblings

Mingfeng Li, Kenneth Lay, Andreas Zimmer, Kristin Technau-Hafsi, Jasmine Wong, Antonia Reimer-Taschenbrecker, Jan Rohr, Ebtesam Abdalla, Judith Fischer, Bruno Reversade, Cristina Has

Background: Trio exome sequencing study identified a previously unreported NLRP1 variant resulting in a p.Leu813Pro substitution of the LRR domain of NLRP1. This homozygous mutation was shared by two sisters with different clinical presentation: the younger sister had generalized inflammatory nodules with keratotic plugs, clinically resembling multiple keratoacanthomas, while the older had manifestations of familial keratosis lichenoides chronica.

Objectives: To analyse the consequences of this NLRP1 variant in two siblings with different clinical spectrum of severity.

Methods: To demonstrate the pathogenicity, p.Leu813Pro was recombinantly expressed and its effect on inflammasome assembly was assessed. Exome sequencing and RNA-Seq were performed to identify factors with potentially modifying effects on the severity of the skin manifestation between each sibling.

Results: The variant p.Leu813Pro triggered the activation of the NLRP1 inflammasome leading to ASC speck formation and IL-1β release. The more severely affected sister had several additional genomic variants associated with atopy and psoriasis that were not present in her sibling. IL-5 and IL-17 emerged as dominant cytokines driving prominent inflammation in the skin of the severely affected sibling.

Conclusions: To the best of our knowledge, this is the first report of a NLRP1 variant that leads to different clinical spectrum of severity within the same sibship. IL-5 and IL-17 were the main cytokines expressed in the inflammatory lesions of the severely affected patient and might be regarded as disease modifying factors and therefore may be considered as therapeutic targets.

Nature Communications

INTS13 variants causing a recessive developmental ciliopathy disrupt assembly of the Integrator complex

Lauren G. Mascibroda, Mohammad Shboul, Nathan D. Elrod, Laurence Colleaux, Hanan Hamamy, Kai-Lieh Huang, Natoya Peart, Moirangthem Kiran Singh, Hane Lee, Barry Merriman, Jeanne N. Jodoin, Poojitha Sitaram, Laura A. Lee, Raja Fathalla, Baeth Al-Rawashdeh, Osama Ababneh, Mohammad El-Khateeb, Nathalie Escande-Beillard, Stanley F. Nelson, Yixuan Wu, Liang Tong, Linda J. Kenney, Sudipto Roy, William K. Russell, Jeanne Amiel, Bruno Reversade & Eric J. Wagner  

Oral-facial-digital (OFD) syndromes are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. Mutations within 12 cilia-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline variants in INTS13, a subunit of the Integrator complex. This multiprotein complex associates with RNA Polymerase II and cleaves nascent RNA to modulate gene expression. We determined that INTS13 utilizes its C-terminus to bind the Integrator cleavage module, which is disrupted by the identified germline variants p.S652L and p.K668Nfs*9. Depletion of INTS13 disrupts ciliogenesis in human cultured cells and causes dysregulation of a broad collection of ciliary genes. Accordingly, its knockdown in Xenopus embryos leads to motile cilia anomalies. Altogether, we show that mutations in INTS13 cause an autosomal recessive ciliopathy, which reveals key interactions between components of the Integrator complex.

Science Immunology

DPP9 deficiency: An inflammasomopathy that can be rescued 

by lowering NLRP1/IL-1 signaling

Cassandra R Harapas 1 2, Kim S Robinson 3 4, Kenneth Lay 5, Jasmine Wong 5, Ricardo Moreno Traspas 5, Nasrin Nabavizadeh 5, Annick Rass-Rothschild 6, Bertrand Boisson 7 8 9, Scott B Drutman 7, Pawat Laohamonthonkul 1 2, Devon Bonner 10, Jingwei Rachel Xiong 3, Mark D Gorrell 11, Sophia Davidson 1 2, Chien-Hsiung Yu 1 2, Mark D Fleming 12, Jonas Gudera 13 14, Jerry Stein 15, Miriam Ben-Harosh 16, Emily Groopman 17 18, Akiko Shimamura 13, Hannah Tamary 15, Hülya Kayserili 19, Nevin Hatipoğlu , Jean-Laurent Casanova, Jonathan A Bernstein, Franklin L Zhong , Seth L Masters , Bruno Reversade 

Dipeptidyl peptidase 9 (DPP9) is a direct inhibitor of NLRP1, but how it affects inflammasome regulation in vivo is not yet established. Here, we report three families with immune-associated defects, poor growth, pancytopenia, and skin pigmentation abnormalities that segregate with biallelic DPP9 rare variants. Using patient-derived primary cells and biochemical assays, these variants were shown to behave as hypomorphic or knockout alleles that failed to repress NLRP1. The removal of a single copy of Nlrp1a/b/c, Asc, Gsdmd, or Il-1r, but not Il-18, was sufficient to rescue the lethality of Dpp9 mutant neonates in mice. Similarly, dpp9 deficiency was partially rescued by the inactivation of asc, an obligate downstream adapter of the NLRP1 inflammasome, in zebrafish. These experiments suggest that the deleterious consequences of DPP9 deficiency were mostly driven by the aberrant activation of the canonical NLRP1 inflammasome and IL-1β signaling. Collectively, our results delineate a Mendelian disorder of DPP9 deficiency driven by increased NLRP1 activity as demonstrated in patient cells and in two animal models of the disease.

Nature Genetics

Loss of FOCAD, operating via the SKI messenger RNA surveillance pathway, causes a pediatric syndrome with liver cirrhosis

Ricardo Moreno Traspas 1 2, Tze Shin Teoh 3 4, Pui-Mun Wong 3, Michael Maier 3, Crystal Y Chia 3, Kenneth Lay 3, Nur Ain Ali 3, Austin Larson 5, Fuad Al Mutairi 6 7, Nouriya Abbas Al-Sannaa 8, Eissa Ali Faqeih 9, Majid Alfadhel 6 10, Huma Arshad Cheema 11, Juliette Dupont 12, Stéphane Bézieau 13, Bertrand Isidor 13, Dorrain Yanwen Low 14, Yulan Wang 14, Grace Tan 4, Poh San Lai 4, Hugues Piloquet 15, Madeleine Joubert 16, Hulya Kayserili 17, Kimberly A Kripps 18, Shareef A Nahas 19, Eric P Wartchow 20, Mikako Warren 21, Gandham SriLakshmi Bhavani 22, Majed Dasouki 23, Renata Sandoval 24, Elisa Carvalho 25, Luiza Ramos 26, Gilda Porta 27, Bin Wu 28 29, Harsha Prasada Lashkari 30 31, Badr AlSaleem 32, Raeda M BaAbbad 32, Anabela Natália Abreu Ferrão 33, Vasiliki Karageorgou 34, Natalia Ordonez-Herrera 34, Suliman Khan 34, Peter Bauer 34, Benjamin Cogne 13, Aida M Bertoli-Avella 34, Marie Vincent, Katta Mohan Girisha, Bruno Reversade 

Cirrhosis is usually a late-onset and life-threatening disease characterized by fibrotic scarring and inflammation that disrupts liver architecture and function. While it is typically the result of alcoholism or hepatitis viral infection in adults, its etiology in infants is much less understood. In this study, we report 14 children from ten unrelated families presenting with a syndromic form of pediatric liver cirrhosis. By genome/exome sequencing, we found recessive variants in FOCAD segregating with the disease. Zebrafish lacking focad phenocopied the human disease, revealing a signature of altered messenger RNA (mRNA) degradation processes in the liver. Using patient's primary cells and CRISPR-Cas9-mediated inactivation in human hepatic cell lines, we found that FOCAD deficiency compromises the SKI mRNA surveillance pathway by reducing the levels of the RNA helicase SKIC2 and its cofactor SKIC3. FOCAD knockout hepatocytes exhibited lowered albumin expression and signs of persistent injury accompanied by CCL2 overproduction. Our results reveal the importance of FOCAD in maintaining liver homeostasis and disclose a possible therapeutic intervention point via inhibition of the CCL2/CCR2 signaling axis.

Nature Communications

HOX epimutations driven by maternal SMCHD1/LRIF1 haploinsufficiency trigger homeotic transformations in genetically wildtype offspring

Shifeng Xue, Thanh Thao Nguyen Ly, Raunak S Vijayakar, Jingyi Chen, Joel Ng, Ajay S Mathuru, Frederique Magdinier, Bruno Reversade  

The body plan of animals is laid out by an evolutionary-conserved HOX code which is colinearly transcribed after zygotic genome activation (ZGA). Here we report that SMCHD1, a chromatin-modifying enzyme needed for X-inactivation in mammals, is maternally required for timely HOX expression. Using zebrafish and mouse Smchd1 knockout animals, we demonstrate that Smchd1 haplo-insufficiency brings about precocious and ectopic HOX transcription during oogenesis and embryogenesis. Unexpectedly, wild-type offspring born to heterozygous knockout zebrafish smchd1 mothers exhibited patent vertebrate patterning defects. The loss of maternal Smchd1 was accompanied by HOX epi-mutations driven by aberrant DNA methylation. We further show that this regulation is mediated by Lrif1, a direct interacting partner of Smchd1, whose knockout in zebrafish phenocopies that of Smchd1. Rather than being a short-lived maternal effect, HOX mis-regulation is stably inherited through cell divisions and persists in cultured fibroblasts derived from FSHD2 patients haploinsufficient for SMCHD1. We conclude that maternal SMCHD1/LRIF1 sets up an epigenetic state in the HOX loci that can only be reset in the germline. Such an unusual inter-generational inheritance, whereby a phenotype can be one generation removed from its genotype, casts a new light on how unresolved Mendelian diseases may be interpreted.

Molecular Cell

Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells

Planès R, Pinilla M, Santoni K, Hessel A, Passemar C, Lay K, Paillette P, Valadão AC, Robinson KS, Bastard P, Lam N, Fadrique R, Rossi I, Pericat D, Bagayoko S, Leon-Icaza SA, Rombouts Y, Perouzel E, Tiraby M; COVID Human Genetic Effort, Zhang Q, Cicuta P, Jouanguy E, Neyrolles O, Bryant CE, Floto AR, Goujon C, Lei FZ, Martin-Blondel G, Silva S, Casanova JL, Cougoule C, Reversade B, Marcoux J, Ravet E, Meunier E.  

Inflammation observed in SARS-CoV-2-infected patients suggests that inflammasomes, proinflammatory intracellular complexes, regulate various steps of infection. Lung epithelial cells express inflammasome-forming sensors and constitute the primary entry door of SARS-CoV-2. Here, we describe that the NLRP1 inflammasome detects SARS-CoV-2 infection in human lung epithelial cells. Specifically, human NLRP1 is cleaved at the Q333 site by multiple coronavirus 3CL proteases, which triggers inflammasome assembly and cell death and limits the production of infectious viral particles. Analysis of NLRP1-associated pathways unveils that 3CL proteases also inactivate the pyroptosis executioner Gasdermin D (GSDMD). Subsequently, caspase-3 and GSDME promote alternative cell pyroptosis. Finally, analysis of pyroptosis markers in plasma from COVID-19 patients with characterized severe pneumonia due to autoantibodies against, or inborn errors of, type I interferons (IFNs) highlights GSDME/caspase-3 as potential markers of disease severity. Overall, our findings identify NLRP1 as a sensor of SARS-CoV-2 infection in lung epithelia.

Nature Methods

Direct identification of A-to-I editing sites with nanopore native RNA sequencing

Nguyen TA, Heng JWJ, Kaewsapsak P, Kok EPL, Stanojević D, Liu H, Cardilla A, Praditya A, Yi Z, Lin M, Aw JGA, Ho YY, Peh KLE, Wang Y, Zhong Q, Heraud-Farlow J, Xue S, Reversade B, Walkley C, Ho YS, Šikić M, Wan Y, Tan MH.  

Inosine is a prevalent RNA modification in animals and is formed when an adenosine is deaminated by the ADAR family of enzymes. Traditionally, inosines are identified indirectly as variants from Illumina RNA-sequencing data because they are interpreted as guanosines by cellular machineries. However, this indirect method performs poorly in protein-coding regions where exons are typically short, in non-model organisms with sparsely annotated single-nucleotide polymorphisms, or in disease contexts where unknown DNA mutations are pervasive. Here, we show that Oxford Nanopore direct RNA sequencing can be used to identify inosine-containing sites in native transcriptomes with high accuracy. We trained convolutional neural network models to distinguish inosine from adenosine and guanosine, and to estimate the modification rate at each editing site. Furthermore, we demonstrated their utility on the transcriptomes of human, mouse and Xenopus. Our approach expands the toolkit for studying adenosine-to-inosine editing and can be further extended to investigate other RNA modifications.

Human Molecular Genetics

RABENOSYN separation-of-function mutations uncouple endosomal recycling from lysosomal degradation, causing a distinct Mendelian Disorder

Paul F, Ng C, Sahari UBM, Nafissi S, Nilipoor Y, Tavasoli AR, Bonnard C, Wong PM, Nabavizadeh N, Altunoğlu U, Estiar MA, Majoie CB, Lee H, Nelson SF, Gan-Or Z, Rouleau GA, Van Veldhoven PP, Massie R, Hennekam RC, Kariminejad A, Reversade B.

Rabenosyn (RBSN) is a conserved endosomal protein necessary for regulating internalized cargo. Here, we present clinical, genetic, cellular and biochemical evidence that two distinct RBSN missense variants are responsible for a novel Mendelian disorder consisting of progressive muscle weakness, facial dysmorphisms, ophthalmoplegia and intellectual disability. Using exome sequencing, we identified recessively-acting germline alleles p.Arg180Gly and p.Gly183Arg which are both situated in the FYVE domain of RBSN. We find that these variants abrogate binding to its cognate substrate PI3P and thus prevent its translocation to early endosomes. Although the endosomal recycling pathway was unaltered, mutant p.Gly183Arg patient fibroblasts exhibit accumulation of cargo tagged for lysosomal degradation. Our results suggest that these variants are separation-of-function alleles, which cause a delay in endosomal maturation without affecting cargo recycling. We conclude that distinct germline mutations in RBSN cause non-overlapping phenotypes with specific and discrete endolysosomal cellular defects.

Developmental Cell

RSPONDIN2 + mesenchymal cells form the bud tip progenitor niche during human lung development

Hein RFC, Wu JH, Holloway EM, Frum T, Conchola AS, Tsai YH, Wu A, Fine AS, Miller AJ, Szenker-Ravi E, Yan KS, Kuo CJ, Glass I, Reversade B, Spence JR.

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2+ mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency.

Stem Cell Reports

Leveraging interacting signaling pathways to robustly improve the quality and yield of human pluripotent stem cell-derived hepatoblasts and hepatocytes

Claudia Raggi 1, Marie-Agnès M'Callum 2, Quang Toan Pham 2, Perrine Gaub 3, Silvia Selleri 2, Nissan Vida Baratang 4, Chenicka Lyn Mangahas 2, Gaël Cagnone 5, Bruno Reversade 6, Jean-Sébastien Joyal 7, Massimiliano Paganelli  

Pluripotent stem cell (PSC)-derived hepatocyte-like cells (HLCs) have shown great potential as an alternative to primary human hepatocytes (PHHs) for in vitro modeling. Several differentiation protocols have been described to direct PSCs toward the hepatic fate. Here, by leveraging recent knowledge of the signaling pathways involved in liver development, we describe a robust, scalable protocol that allowed us to consistently generate high-quality bipotent human hepatoblasts and HLCs from both embryonic stem cells and induced PSC (iPSCs). Although not yet fully mature, such HLCs were more similar to adult PHHs than were cells obtained with previously described protocols, showing good potential as a physiologically representative alternative to PHHs for in vitro modeling. PSC-derived hepatoblasts effectively generated with this protocol could differentiate into mature hepatocytes and cholangiocytes within syngeneic liver organoids, thus opening the way for representative human 3D in vitro modeling of liver development and pathophysiology.

Brain Pathology

Ermin deficiency leads to compromised myelin, inflammatory milieu, and susceptibility to demyelinating insult

Amin Ziaei 1 2, Marta Garcia-Miralles 1, Carola I Radulescu 1, Harwin Sidik 1, Aymeric Silvin 3, Han-Gyu Bae 4 5, Carine Bonnard 6, Nur Amirah Binte Mohammad Yusof 1, Costanza Ferrari Bardile 1 7, Liang Juin Tan 1, Alvin Yu Jin Ng 4, Sumanty Tohari 4, Leila Dehghani 8, Lily Henry 1, Xin Yi Yeo 4, Sejin Lee 4, Byrappa Venkatesh 4 9, Sarah R Langley 10, Vahid Shaygannejad 8, Bruno Reversade , Sangyong Jung , Florent Ginhoux , Mahmoud A Pouladi 

Ermin is an actin-binding protein found almost exclusively in the central nervous system (CNS) as a component of myelin sheaths. Although Ermin has been predicted to play a role in the formation and stability of myelin sheaths, this has not been directly examined in vivo. Here, we show that Ermin is essential for myelin sheath integrity and normal saltatory conduction. Loss of Ermin in mice caused de-compacted and fragmented myelin sheaths and led to slower conduction along with progressive neurological deficits. RNA sequencing of the corpus callosum, the largest white matter structure in the CNS, pointed to inflammatory activation in aged Ermin-deficient mice, which was corroborated by increased levels of microgliosis and astrogliosis. The inflammatory milieu and myelin abnormalities were further associated with increased susceptibility to immune-mediated demyelination insult in Ermin knockout mice. Supporting a possible role of Ermin deficiency in inflammatory white matter disorders, a rare inactivating mutation in the ERMN gene was identified in multiple sclerosis patients. Our findings demonstrate a critical role for Ermin in maintaining myelin integrity. Given its near-exclusive expression in myelinating oligodendrocytes, Ermin deficiency represents a compelling "inside-out" model of inflammatory dysmyelination and may offer a new paradigm for the development of myelin stability-targeted therapies.

Frontiers in Immunology

C10orf99/GPR15L Regulates Proinflammatory Response of Keratinocytes and Barrier Formation of the Skin

Teruki Dainichi 1 2, Yuri Nakano 2, Hiromi Doi 2, Satoshi Nakamizo 2 3, Saeko Nakajima 2 4, Reiko Matsumoto 2, Thomas Farkas 5, Pui Mun Wong 6, Vipin Narang 7, Ricardo Moreno Traspas 6, Eiryo Kawakami 8 9, Emma Guttman-Yassky 10, Oliver Dreesen 3, Thomas Litman 5, Bruno Reversade 6, Kenji Kabashima  

The epidermis, outermost layer of the skin, forms a barrier and is involved in innate and adaptive immunity in an organism. Keratinocytes participate in all these three protective processes. However, a regulator of keratinocyte protective responses against external dangers and stresses remains elusive. We found that upregulation of the orphan gene 2610528A11Rik was a common factor in the skin of mice with several types of inflammation. In the human epidermis, peptide expression of G protein-coupled receptor 15 ligand (GPR15L), encoded by the human ortholog C10orf99, was highly induced in the lesional skin of patients with atopic dermatitis or psoriasis. C10orf99 gene transfection into normal human epidermal keratinocytes (NHEKs) induced the expression of inflammatory mediators and reduced the expression of barrier-related genes. Gene ontology analyses showed its association with translation, mitogen-activated protein kinase (MAPK), mitochondria, and lipid metabolism. Treatment with GPR15L reduced the expression levels of filaggrin and loricrin in human keratinocyte 3D cultures. Instead, their expression levels in mouse primary cultured keratinocytes did not show significant differences between the wild-type and 2610528A11Rik deficient keratinocytes. Lipopolysaccharide-induced expression of Il1b and Il6 was less in 2610528A11Rik deficient mouse keratinocytes than in wild-type, and imiquimod-induced psoriatic dermatitis was blunted in 2610528A11Rik deficient mice. Furthermore, repetitive subcutaneous injection of GPR15L in mouse ears induced skin inflammation in a dose-dependent manner. These results suggest that C10orf99/GPR15L is a primary inducible regulator that reduces the barrier formation and induces the inflammatory response of keratinocytes.

American Journal of Medical Genetics A

Huriez syndrome: Additional pathogenic variants supporting allelism to SMARCAD syndrome

Abigail Y T Loh 1 2, Sanja Špoljar 3, Granville Y W Neo 1, Nathalie Escande-Beillard 1 2 4, Marc Leushacke 5, Monique N H Luijten 2, Byrappa Venkatesh 2, Carine Bonnard 5, Maurice A M van Steensel 5 6, Henning Hamm 7, Andrew Carmichael 8, Neil Rajan 9, Thomas J Carney 2 6, Bruno Reversade 

Huriez syndrome (HRZ, OMIM181600) is a rare genodermatosis characterized by scleroatrophic hands and feet, hypoplastic nails, palmoplantar keratoderma, and predisposition to cutaneous squamous cell carcinoma (cSCC). We report herein three HRZ families from Croatia, the Netherlands, and Germany. Deep sequencing followed by Sanger validation, confirmed the presence of germline causative SMARCAD1 heterozygous pathogenic variants. All seven HRZ patients displayed hypohidrosis, adermatoglyphia, and one patient developed cSCC at 32 years of age. Two novel monoallelic germline mutations were identified which are predicted to disrupt the first exon-intron boundary of the skin-specific SMARCAD1 isoform. On the basis of phenotypic and genotypic convergence with Adermatoglyphia (OMIM136000) and Basan syndrome (OMIM129200), our results lend credence to the notion that these three Mendelian disorders are allelic. We propose adding Huriez syndrome to the previously suggested SMARCAD syndrome designation, which was originally invoked to describe the spectrum of monogenic disorders between Adermatoglyphia and Basan syndrome.

Nature Genetics

Discovery of a genetic module essential for assigning left-right asymmetry in humans and ancestral vertebrates

Emmanuelle Szenker-Ravi, Tim Ott, Muznah Khatoo, Anne Moreau de Bellaing 4, Wei Xuan Goh 3, Yan Ling Chong 5 6, Anja Beckers 7 8, Darshini Kannesan 3, Guillaume Louvel 9 10, Priyanka Anujan 5 11, Vydianathan Ravi 5, Carine Bonnard 12, Sébastien Moutton 13, Patric Schoen 14, Mélanie Fradin 15, Estelle Colin 16, André Megarbane 17 18, Linda Daou 19, Ghassan Chehab 19 20, Sylvie Di Filippo 21, Caroline Rooryck 22, Jean-François Deleuze 23, Anne Boland 23, Nicolas Arribard 24, Rukiye Eker 25, Sumanty Tohari 5, Alvin Yu-Jin Ng 26, Marlène Rio, Chun Teck Lim 29 30, Birgit Eisenhaber, Frank Eisenhaber, Byrappa Venkatesh 5 33, Jeanne Amiel 27 34, Hugues Roest Crollius 9, Christopher T Gordon 34, Achim Gossler, Sudipto Roy, Tania Attie-Bitach, Martin Blum, Patrice Bouvagnet , Bruno Reversade

The vertebrate left-right axis is specified during embryogenesis by a transient organ: the left-right organizer (LRO). Species including fish, amphibians, rodents and humans deploy motile cilia in the LRO to break bilateral symmetry, while reptiles, birds, even-toed mammals and cetaceans are believed to have LROs without motile cilia. We searched for genes whose loss during vertebrate evolution follows this pattern and identified five genes encoding extracellular proteins, including a putative protease with hitherto unknown functions that we named ciliated left-right organizer metallopeptide (CIROP). Here, we show that CIROP is specifically expressed in ciliated LROs. In zebrafish and Xenopus, CIROP is required solely on the left side, downstream of the leftward flow, but upstream of DAND5, the first asymmetrically expressed gene. We further ascertained 21 human patients with loss-of-function CIROP mutations presenting with recessive situs anomalies. Our findings posit the existence of an ancestral genetic module that has twice disappeared during vertebrate evolution but remains essential for distinguishing left from right in humans.

2020

Human Mutation

Next-generation sequencing in a series of 80 fetuses with complex cardiac malformations and/or heterotaxy

Hui Liu , Anna-Gaëlle Giguet-Valard , Thomas Simonet , Emmanuelle Szenker-Ravi , Laetitia Lambert , Catherine Vincent-Delorme , Sophie Scheidecker , Mélanie Fradin , Fanny Morice-Picard , Sophie Naudion , Viorica Ciorna-Monferrato , Estelle Colin , Florence Fellmann , Sophie Blesson , Pierre-Simon Jouk Christine Francannet, Florence Petit , Sébastien Moutton , Daphné Lehalle , Nicolas Chassaing, Loubna El Zein , Anne Bazin , Claire Bénéteau , Tania Attié-Bitach , Sylvie M Hanu , Marie-Pierre Brechard , Jean Chiesa , Laurent Pasquier , Caroline Rooryck-Thambo , Lionel Van Maldergem , Christelle Cabrol , Salima El Chehadeh , Alexandre Vasiljevic , Bertrand Isidor , Carine Abel , Julien Thevenon , Sylvie Di Filippo , Adeline Vigouroux-Castera , Jocelyne Attia , Chloé Quelin , Sylvie Odent , Juliette Piard , Fabienne Giuliano , Audrey Putoux , Philippe Khau Van Kien , Catherine Yardin , Renaud Touraine , Bruno Reversade , Patrice Bouvagnet 

Herein, we report the screening of a large panel of genes in a series of 80 fetuses with congenital heart defects (CHDs) and/or heterotaxy and no cytogenetic anomalies. There were 49 males (61%/39%), with a family history in 28 cases (35%) and no parental consanguinity in 77 cases (96%). All fetuses had complex CHD except one who had heterotaxy and midline anomalies while 52 cases (65%) had heterotaxy in addition to CHD. Altogether, 29 cases (36%) had extracardiac and extra-heterotaxy anomalies. A pathogenic variant was found in 10/80 (12.5%) cases with a higher percentage in the heterotaxy group (8/52 cases, 15%) compared with the non-heterotaxy group (2/28 cases, 7%), and in 3 cases with extracardiac and extra-heterotaxy anomalies (3/29, 10%). The inheritance was recessive in six genes (DNAI1, GDF1, MMP21, MYH6, NEK8, and ZIC3) and dominant in two genes (SHH and TAB2). A homozygous pathogenic variant was found in three cases including only one case with known consanguinity. In conclusion, after removing fetuses with cytogenetic anomalies, next-generation sequencing discovered a causal variant in 12.5% of fetal cases with CHD and/or heterotaxy. Genetic counseling for future pregnancies was greatly improved. Surprisingly, unexpected consanguinity accounts for 20% of cases with identified pathogenic variants.

Science

Enteroviral 3C protease activates the human NLRP1 inflammasome in airway epithelia

Kim S. Robinson, Daniel Eng Thiam Teo, Kai Sen Tan, Gee Ann Toh, Hsiao Hui Ong, Chrissie Kaishi Lim, Kenneth Lay, Bijin Veonice Au, View, Tian Sheng Lew, Justin Jang Hann Chu, Vincent Tak Kwong Chow, De Yun Wang, Franklin L. Zhong, Bruno Reversade.

Immune sensor proteins are critical to the function of the human innate immune system. The full repertoire of cognate triggers for human immune sensors is not fully understood. Here, we report that human NLRP1 is activated by 3C proteases (3Cpros) of enteroviruses, such as human rhinovirus (HRV). 3Cpros directly cleave human NLRP1 at a single site between Glu130 and Gly131. This cleavage triggers N-glycine–mediated degradation of the autoinhibitory NLRP1 N-terminal fragment via the cullinZER1/ZYG11B complex, which liberates the activating C-terminal fragment. Infection of primary human airway epithelial cells by live human HRV triggers NLRP1-dependent inflammasome activation and IL-18 secretion. Our findings establish 3Cpros as a pathogen-derived trigger for the human NLRP1 inflammasome and suggest that NLRP1 may contribute to inflammatory diseases of the airway.

Nature Communications

Loss of MTX2 causes Mandibuloacral Dysplasia and links mitochondrial dysfunction to altered nuclear morphology

Sahar Elouej, Karim Harhouri, Morgane Lemao, Genevieve Baujat, Sheela Nampoothiri, Hulya Kayserili, Nihal Al Menabawy, Laila Selim, Arianne LLamos Paneque, Christian Kubisch, Davor Lessel, Robert Rubinsztajn, Chayki Charar, Catherine Bartoli, Coraline Airault, Jean-François Deleuze, Agnes Rotig, Peter Bauer, Catarina Pereira, Abigail Loh, Nathalie Escande-Beillard, Antoine Muchir, Lisa Martino, Yosef Gruenbaum, Song-Hua Lee, Philippe Manivet, Guy Lenaers, Bruno Reversade, Nicolas Levy,  Annachiara De Sandre-Giovannoli.

Mandibuloacral dysplasia syndromes are mainly due to recessive LMNA or ZMPSTE24 mutations, with cardinal nuclear morphological abnormalities and dysfunction. We report five homozygous null mutations in MTX2, encoding Metaxin-2 (MTX2), an outer mitochondrial membrane protein, in patients presenting with a severe laminopathy-like mandibuloacral dysplasia characterized by growth retardation, bone resorption, arterial calcification, renal glomerulosclerosis and severe hypertension. Loss of MTX2 in patients’ primary fibroblasts leads to loss of Metaxin-1 (MTX1) and mitochondrial dysfunction, including network fragmentation and oxidative phosphorylation impairment. Furthermore, patients’ fibroblasts are resistant to induced apoptosis, leading to increased cell senescence and mitophagy and reduced proliferation. Interestingly, secondary nuclear morphological defects are observed in both MTX2-mutant fibroblasts and mtx-2-depleted C. elegans. We thus report the identification of a severe premature aging syndrome revealing an unsuspected link between mitochondrial composition and function and nuclear morphology, establishing a pathophysiological link with premature aging laminopathies and likely explaining common clinical features. 

Journal of Experimental Medicine

A Loss-of-function NUAK2 Mutation in Humans Causes Anencephaly Due to Impaired Hippo-YAP Signaling

Carine Bonnard, Naveenan Navaratnam, Kakaly Ghosh, Puck Wee Chan, Thong Teck Tan, Oz Pomp, Alvin Yu, Jin Ng, Sumanty Tohari, Rishita Changede, David Carling, Byrappa Venkatesh, Umut Altunoglu, Hülya Kayserili, Bruno Reversade.

Failure of neural tube closure during embryonic development can result in anencephaly, one of the most common birth defects in humans. A family with recurrent anencephalic fetuses was investigated to understand its etiology and pathogenesis. Exome sequencing revealed a recessive germline 21 bp in-frame deletion in NUAK2 segregating with the disease. In vitro kinase assays demonstrated that the 7 amino-acid truncation in NUAK2, a serine/threonine kinase, completely abrogated its catalytic activity. Patient-derived disease models including neural progenitor cells and cerebral organoids showed that loss of NUAK2 activity led to decreased Hippo signaling via cytoplasmic YAP retention. In neural tube-like structures, endogenous NUAK2 co-localized apically with the actomyosin network, which was disrupted in patient’s cells causing impaired nucleokinesis and apical constriction. Our results establish NUAK2 as an indispensable kinase for brain development in humans and suggest that a NUAK2-Hippo signaling axis regulates cytoskeletal processes that govern cell shape during neural tube closure.

Journal of Clinical Investigation 

Specific Missense Mutation in NFKBIA underlies Severe Immunodeficiency, Hyper-activation IL-1beta responses, and IL-1-Mediated Hepatic Disease.

Enrica Ee Kar Tan, Richard Hopkins, Chrissie K Lim, Saumya Jamuar, Christina Ong, Koh Cheng Thoon, Mark Ja Koh, Eun Myoung Shin, Derrick Wen Quan Lian, Madhushanee Weerasooriya, Christopher Zw Lee, Andreas Alvin Purnomo Soetedjo, Chang Siang Lim, Veronica B Au, W M Edmond Chua, Hui Yin Lee, Leigh Ann Jones, Sharmy Jennifer James, Nivashini Kaliaperumal, Jeffrey Kwok, Ee Shien Tan, Biju Thomas, Lena Ho, Lynn Wu, Anna-Marie Fairhurst, Florent Ginhoux, Adrian Kk Teo, Yongliang Zhang, Kok Haur Ong, Weimiao Yu, Byrappa Venkatesh, Vinay Tergaonkar, Bruno Reversade, Keh-Chuang Chin, Ah Moy Tan, Woei Kang Liew, John E Connolly.

Although IKK-β has previously been shown as a negative regulator of IL-1β secretion in mice, this role has not been demonstrated in humans. Genetic studies of NF-κB signalling in humans with inherited diseases of the immune system have not demonstrated the relevance of the NF-κB pathway in suppression of IL-1β expression. Here, we report an infant displaying clinical pathology comprising neutrophil-mediated auto-inflammation and recurrent bacterial infections. Whole-exome sequencing revealed a de novo heterozygous missense mutation in NFKBIA, resulting in a L34P IκBα variant, that severely repressed NF-κB activation and downstream cytokine production. Paradoxically, IL-1β secretion was elevated in the patient's stimulated leukocytes, in her induced-pluripotent stem cell-derived macrophages, and in murine bone marrow-derived macrophages containing the L34P mutation. The patient's hyper-IL-1β secretion correlated with activated neutrophilia and liver fibrosis with neutrophil accumulation. Hematopoietic stem cell transplantation reversed neutrophilia, restored a resting state in neutrophils, and normalized IL-1β release from stimulated leukocytes. Additional therapeutic blockade of IL-1 ameliorated liver damage, whilst decreasing neutrophil activation and associated IL-1β secretion. Our studies reveal a previously unrecognized role of human IκBα as an essential regulator of canonical NF-κB signalling in the prevention of neutrophilic-dependent auto-inflammatory diseases. We showed that IκBα controls IL-1β secretion through a mechanism of self-limiting post-transcriptional regulation. These findings also highlight a therapeutic potential for IL-1 inhibitors to treat complications arising from systemic NF-κB inhibition.

eLiFE 

R-spondin signalling is essential for the maintenance and differentiation of mouse nephron progenitors

Valerie Vidal , Fariba Jian Motamedi, Samah Rekima, Elodie P Gregoire, Emmanuelle Szenker-Ravi, Marc Leushacke, Bruno Reversade, Marie-Christine Chaboissier, Andreas Schedl

During kidney development, WNT/b-catenin signalling has to be tightly controlled to ensure proliferation and differentiation of nephron progenitor cells. Here we show in mice that the signalling molecules RSPO1 and RSPO3 act in a functionally redundant manner to permit WNT/b-catenin signalling and their genetic deletion leads to a rapid decline of nephron progenitors. By contrast, tissue specific deletion in cap mesenchymal cells abolishes mesenchyme to epithelial transition (MET) that is linked to a loss of Bmp7 expression, absence of SMAD1/5 phosphorylation and a concomitant failure to activate Lef1, Fgf8 and Wnt4, thus explaining the observed phenotype on a molecular level. Surprisingly, the full knockout of LGR4/5/6, the cognate receptors of R-spondins, only mildly affects progenitor numbers, but does not interfere with MET. Taken together our data demonstrate key roles for R-spondins in permitting stem cell maintenance and differentiation and reveal Lgr-dependent and independent functions for these ligands during kidney formation.

Neuron

Loss of PYCR2 causes Neurodegeneration by increasing Cerebral Glycine levels via SHMT2

Nathalie Escande-Beillard, Abigail Loh, Sahar N. Saleem, Kohei Kanata, Yui Hashimoto, Umut Altunoglu, Artina Metoska, Joanes Grandjean, Fui Mee Ng, Oz Pomp, Nithya Baburajendran, Joyner Wong, Jeffrey Hill, Emmanuel Beillard, Patrick Cozzone, Maha Zaki, Hülya Kayserili, Hiroshi Hamada, Hidetaka Shiratori, Bruno Reversade 

Patients lacking PYCR2, a mitochondrial enzyme that synthesizes proline, display postnatal degenerative microcephaly with hypomyelination. Here we report the crystal structure of the PYCR2 apo-enzyme and show that a novel germline p.Gly249Val mutation lies at the dimer interface and lowers its enzymatic activity. We find that knocking out Pycr2 in mice phenocopies the human disorder and depletes PYCR1 levels in neural lineages. In situ quantification of neurotransmitters in the brains of PYCR2 mutant mice and patients revealed a signature of encephalopathy driven by excessive cerebral glycine. Mechanistically, we demonstrate that loss of PYCR2 upregulates SHMT2, which is responsible for glycine synthesis. This hyperglycemia could be partially reversed by SHMT2 knockdown, which rescued the axonal beading and neurite lengths of cultured Pycr2 knockout neurons. Our findings identify the glycine metabolic pathway as a possible intervention point to alleviate the neurological symptoms of PYCR2-mutant patients.

Nature Communications 

Mitochondrial peptide BRAWNIN is essential for vertebrate respiratory complex III assembly

Zhang S, Reljić B, Liang C, Kerouanton B, Francisco JC, Peh JH, Mary C, Jagannathan NS, Olexiouk V, Tang C, Fidelito G, Nama S, Cheng RK, Wee CL, Wang LC, Duek Roggli P, Sampath P, Lane L, Petretto E, Sobota RM, Jesuthasan S, Tucker-Kellogg L, Reversade B, Menschaert G, Sun L, Stroud DA, Ho L.

The emergence of small open reading frame (sORF)-encoded peptides (SEPs) is rapidly expanding the known proteome at the lower end of the size distribution. Here, we show that the mitochondrial proteome, particularly the respiratory chain, is enriched for small proteins. Using a prediction and validation pipeline for SEPs, we report the discovery of 16 endogenous nuclear encoded, mitochondrial-localized SEPs (mito-SEPs). Through functional prediction, proteomics, metabolomics and metabolic flux modeling, we demonstrate that BRAWNIN, a 71 a.a. peptide encoded by C12orf73, is essential for respiratory chain complex III (CIII) assembly. In human cells, BRAWNIN is induced by the energy-sensing AMPK pathway, and its depletion impairs mitochondrial ATP production. In zebrafish, Brawnin deletion causes complete CIII loss, resulting in severe growth retardation, lactic acidosis and early death. Our findings demonstrate that BRAWNIN is essential for vertebrate oxidative phosphorylation. We propose that mito-SEPs are an untapped resource for essential regulators of oxidative metabolism.

Nature Communications

Loss-of-function Mutations in UDP-Glucose 6-Dehydrogenase Are a Novel Cause of Recessive Developmental Epileptic Encephalopathy.

Hengel H, Bosso-Lefèvre C, Grady G, Szenker-Ravi E, Li H, Pierce S, Lebigot É, Tan TT, Eio MY, Narayanan G, Utami KH, Yau M, Handal N, Deigendesch W, Keimer R, Marzouqa HM, Gunay-Aygun M, Muriello MJ, Verhelst H, Weckhuysen S, Mahida S, Naidu S, Thomas TG, Lim JY, Tan ES, Haye D, Willemsen MAAP, Oegema R, Mitchell WG, Pierson TM, Andrews MV, Willing MC, Rodan LH, Barakat TS, van Slegtenhorst M, Gavrilova RH, Martinelli D, Gilboa T, Tamim AM, Hashem MO, AlSayed MD, Abdulrahim MM, Al-Owain M, Awaji A, Mahmoud AAH, Faqeih EA, Asmari AA, Algain SM, Jad LA, Aldhalaan HM, Helbig I, Koolen DA, Riess A, Kraegeloh-Mann I, Bauer P, Gulsuner S, Stamberger H, Ng AYJ, Tang S, Tohari S, Keren B, Schultz-Rogers LE, Klee EW, Barresi S, Tartaglia M, Mor-Shaked H, Maddirevula S, Begtrup A, Telegrafi A, Pfundt R, Schüle R, Ciruna B, Bonnard C, Pouladi MA, Stewart JC, Claridge-Chang A, Lefeber DJ, Alkuraya FS, Mathuru AS, Venkatesh B, Barycki JJ, Simpson MA, Jamuar SS, Schöls L, Reversade B.

Developmental epileptic encephalopathies are devastating disorders characterized by intractable epileptic seizures and developmental delay. Here, we report an allelic series of germline recessive mutations in UGDH in 36 cases from 25 families presenting with epileptic encephalopathy with developmental delay and hypotonia. UGDH encodes an oxidoreductase that converts UDP-glucose to UDP-glucuronic acid, a key component of specific proteoglycans and glycolipids. Consistent with being loss-of-function alleles, we show using patients' primary fibroblasts and biochemical assays, that these mutations either impair UGDH stability, oligomerization, or enzymatic activity. In vitro, patient-derived cerebral organoids are smaller with a reduced number of proliferating neuronal progenitors while mutant ugdh zebrafish do not phenocopy the human disease. Our study defines UGDH as a key player for the production of extracellular matrix components that are essential for human brain development. Based on the incidence of variants observed, UGDH mutations are likely to be a frequent cause of recessive epileptic encephalopathy.