Background: DICER1 is the only miRNA biogenesis component associated with an inherited tumor syndrome, featuring multinodular goiter (MNG) and rare pediatric-onset lesions. Other susceptibility genes for familial forms of MNG likely exist. Methods: Whole exome sequencing of a kindred with early-onset MNG and schwannomatosis was followed by investigation of germline pathogenic variants that fully segregated with the disease. Genome wide analyses were performed on 13 tissue samples from familial and non-familial DGCR8-E518K positive tumors, including MNG, schwannomas, papillary thyroid cancers (PTC) and Wilms Tumors. MiRNA profiles of four tissue types were compared, and sequencing of miRNA, pre-miRNA and mRNA was performed in a subset of 9 schwannomas, four of which harbor DGCR8-E518K. Results: We identified c.1552G>A;p.E518K in DGCR8, a microprocessor located in 22q, in the kindred. The variant identified is a somatic hotspot in Wilms Tumors and has been identified in two PTCs. Copy number loss of chromosome 22q, leading to loss of heterozygosity at the DGCR8 locus, was found in all 13 samples harboring c.1552G>A;p.E518K. miRNA profiling of PTC, MNG, schwannomas and Wilms Tumors revealed a common profile among E518K hemizygous tumors. In vitro cleavage demonstrated improper processing of pre-miRNA by DGCR8-E518K. MicroRNA and RNA profiling show that this variant disrupts precursor microRNA production, impacting populations of canonical microRNAs and mirtrons. Conclusions: We identified DGCR8 as the cause of an unreported autosomal dominant mendelian tumor susceptibility syndrome: familial multinodular goiter with schwannomatosis. Funded by CIHR, Compute Canada, Alex’s Lemonade Stand Foundation, and the Mia Neri Foundation for Childhood Cancer.
Barbara Rivera, Javad Nadaf, Somayyeh Fahiminiya, Maria Apellaniz-Ruiz, Avi Saskin, Anne-Sophie Chong, Sahil Sharma, Rabea Wagener, Timothée Revil, Vincenzo Condello, Zineb Harra, Nancy Hamel, Nelly Sabbaghian, Karl Muchantef, Christian Thomas, Leanne de Kock, Marie-Noëlle Hébert-Blouin, Angelia V. Bassenden, Hannah Rabenstein, Ozgur Mete, Ralf Paschke, Marc P. Pusztaszeri, Werner Paulus, Albert Berghuis, Jiannis Ragoussis, Yuri E. Nikiforov, Reiner Siebert, Steffen Albrecht, Robert Turcotte, Martin Hasselblatt, Marc R. Fabian, William D. Foulkes
Immune response to therapeutic enzymes poses a detriment to patient safety and treatment outcome. Enzyme replacement therapy (ERT) is a standard therapeutic option for some types of Mucopolysaccharidoses including Morquio A syndrome caused by GALNS deficiency. Current protocols tolerize patients using cytotoxic immunosuppressives which can cause adverse effects. Here we show development of tolerance in Morquio A mice via oral delivery of peptide or GALNS during ten days prior to ERT. Our results show that using an immunodominant peptide (I10) or the complete enzyme (GALNS) to orally induce tolerance to GALNS prior to ERT, resulted in several improvements to ERT in mice: i) decreased splenocyte proliferation after in-vitro GALNS stimulation; ii) modulation of cytokine secretion profile; iii) decline in GALNS-specific IgG or IgE plasma; iv) decreased GAG storage in liver; and v) fewer circulating immune-complexes in plasma. This model could be extrapolated to other lysosomal storage disorders where immune response hinders ERT.
Angela C. Sosa, Barbara Kariuki, Qi Gan, Alan P. Knutsen, Clifford J. Bellone, Miguel A. Guzmán, Luis A. Barrera, Shunji Tomatsu, Anil K. Chauhan, Eric Armbrecht, Adriana M. Montaño
Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR), with approximately 90% of patients harboring at least one copy of the disease-associated variant F508del. We utilized a yeast phenomic system to identify genetic modifiers of F508del-CFTR biogenesis, from which ribosomal protein L12 (RPL12/uL11) emerged as a molecular target. In the present study, we investigated mechanism(s) by which suppression of RPL12 rescues F508del protein synthesis and activity. Using ribosome profiling, we found that rates of translation initiation and elongation were markedly slowed by RPL12 silencing. However, proteolytic stability and patch-clamp assays revealed RPL12 depletion significantly increased F508del-CFTR steady-state expression, interdomain assembly, and baseline open-channel probability. We next evaluated whether Rpl12-corrected F508del-CFTR could be further enhanced with concomitant pharmacologic repair (e.g., using clinically approved modulators lumacaftor and tezacaftor) and demonstrated additivity of these treatments. Rpl12 knockdown also partially restored maturation of specific CFTR variants in addition to F508del, and WT Cftr biogenesis was enhanced in the pancreas, colon, and ileum of Rpl12 haplosufficient mice. Modulation of ribosome velocity therefore represents a robust method for understanding both CF pathogenesis and therapeutic response.
Kathryn E. Oliver, Robert Rauscher, Marjolein Mijnders, Wei Wang, Matthew J. Wolpert, Jessica Maya, Carleen M. Sabusap, Robert A. Kesterson, Kevin L. Kirk, Andras Rab, Ineke Braakman, Jeong S. Hong, John L. Hartman IV, Zoya Ignatova, Eric J. Sorscher
Gout is caused by deposition of monosodium urate crystals in joints when plasma uric acid levels are chronically elevated beyond the saturation threshold, mostly due to renal underexcretion of uric acid. Although molecular pathways of this underexcretion have been elucidated, its etiology remains mostly unknown. We demonstrate that gout can be caused by a mutation in LDHD within the putative catalytic site of the encoded d-lactate dehydrogenase, resulting in augmented blood levels of d-lactate, a stereoisomer of l-lactate, which is normally present in human blood in miniscule amounts. Consequent excessive renal secretion of d-lactate in exchange for uric acid reabsorption culminated in hyperuricemia and gout. We showed that LDHD expression is enriched in tissues with a high metabolic rate and abundant mitochondria and that d-lactate dehydrogenase resides in the mitochondria of cells overexpressing the human LDHD gene. Notably, the p.R370W mutation had no effect on protein localization. In line with the human phenotype, injection of d-lactate into naive mice resulted in hyperuricemia. Thus, hyperuricemia and gout can result from the accumulation of metabolites whose renal excretion is coupled to uric acid reabsorption.
Max Drabkin, Yuval Yogev, Lior Zeller, Raz Zarivach, Ran Zalk, Daniel Halperin, Ohad Wormser, Evgenia Gurevich, Daniel Landau, Rotem Kadir, Yonatan Perez, Ohad S. Birk
Background: Proteinuria is considered as an unfavorable clinical condition that accelerates renal and cardiovascular disease. However, it is not clear if all forms of proteinuria are damaging. Mutations in CUBN cause Imerslund-Gräsbeck syndrome (IGS) featured by intestinal malabsorption of vitamin B12 and in some cases proteinuria. CUBN encodes for cubilin, an intestinal and proximal tubular uptake receptor containing 27 CUB domains for ligand binding. Methods: We used next-generation sequencing for renal disease genes to genotype cohorts of patients with suspected hereditary renal disease and chronic proteinuria. CUBN variants were analyzed using bioinformatics, structural modeling and epidemiological methods. Results: We identified 39 patients, in whom biallelic pathogenic variants in the CUBN gene are associated with chronic isolated proteinuria with childhood onset. Since the proteinuria displayed a high proportion of albuminuria, glomerular diseases such as steroid-resistant nephrotic syndrome or Alport syndrome were often the primary clinical diagnosis, motivating renal biopsies and proteinuria-lowering treatments. Yet, renal function was normal in all cases. By contrast, we did not find any biallelic pathogenic CUBN variants in patients with reduced renal function or focal segmental glomerulosclerosis. Unlike the more N-terminal IGS mutations, 37 out of the 41 proteinuria-associated CUBN variants led to modifications or truncations after the vitamin B12-binding domain. Finally, we show that four C-terminal CUBN variants are associated with albuminuria and moderately increased GFR in meta-analyses of large population-based cohorts. Conclusions: Collectively, our data suggest an important role for the C-terminal half of cubilin in renal albumin reabsorption. Albuminuria due to reduced cubilin function could be an unexpectedly common benign condition in humans that may not require any proteinuria-lowering treatment or renal biopsies.
Mathilda Bedin, Olivia Boyer, Aude Servais, Yong Li, Laure Villoing-Gaudé, Marie-Josephe Tête, Alexandra Cambier, Julien Hogan, Veronique Baudouin, Saoussen Krid, Albert Bensman, Florie Lammens, Ferielle Louillet, Bruno Ranchin, Cecile Vigneau, Iseline Bouteau, Corinne Isnard-Bagnis, Christoph J. Mache, Tobias Schäfer, Lars Pape, Markus Gödel, Tobias B. Huber, Marcus Benz, Günter Klaus, Matthias Hansen, Kay Latta, Olivier Gribouval, Vincent Morinière, Carole Tournant, Maik Grohmann, Elisa Kuhn, Timo Wagner, Christine Bole-Feysot, Fabienne Jabot-Hanin, Patrick Nitschké, Tarunveer S. Ahluwalia, Anna Köttgen, Christian Brix Folsted Andersen, Carsten Bergmann, Corinne Antignac, Matias Simons
Atrial fibrillation (AF), defined by disorganized atrial cardiac rhythm, is the most prevalent cardiac arrhythmia worldwide. Recent genetic studies have highlighted a major heritable component and identified numerous loci associated with AF risk, including the cardiogenic transcription factor genes TBX5, GATA4, and NKX2-5. We report that Tbx5 and Gata4 interact with opposite signs for atrial rhythm controls compared with cardiac development. Using mouse genetics, we found that AF pathophysiology caused by Tbx5 haploinsufficiency, including atrial arrhythmia susceptibility, prolonged action potential duration, and ectopic cardiomyocyte depolarizations, were all rescued by Gata4 haploinsufficiency. In contrast, Nkx2-5 haploinsufficiency showed no combinatorial effect. The molecular basis of the TBX5/GATA4 interaction included normalization of intra-cardiomyocyte calcium flux and expression of calcium channel genes Atp2a2 and Ryr2. Furthermore, GATA4 and TBX5 showed antagonistic interactions on an Ryr2 enhancer. Atrial rhythm instability caused by Tbx5 haploinsufficiency was rescued by a decreased dose of phospholamban, a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, consistent with a role for decreased sarcoplasmic reticulum calcium flux in Tbx5-dependent AF susceptibility. This work defines a link between Tbx5 dose, sarcoplasmic reticulum calcium flux, and AF propensity. The unexpected interactions between Tbx5 and Gata4 in atrial rhythm control suggest that evaluating specific interactions between genetic risk loci will be necessary for ascertaining personalized risk from genetic association data.
Brigitte Laforest, Wenli Dai, Leonid Tyan, Sonja Lazarevic, Kaitlyn M. Shen, Margaret Gadek, Michael T. Broman, Christopher R. Weber, Ivan P. Moskowitz
Diabetes is a common complication of cystic fibrosis (CF) that affects approximately 20% of adolescents and 40% to 50% of adults with CF. The age-at-onset of CF-related diabetes (marked by clinical diagnosis and treatment initiation) is an important measure of the disease process. DNA variants associated with age-at-onset of CFRD reside in and near SLC26A9. Deep sequencing of the SLC26A9 gene in 762 individuals with CF revealed that two common DNA haplotypes formed by the risk variants account for the association with diabetes (high risk, P-value: 4.34E-3; low risk, P-value: 1.14E-3). Single-cell RNA (scRNA) sequencing indicated that SLC26A9 is predominantly expressed in pancreatic ductal cells, and frequently co-expressed with CFTR along with transcription factors that have binding sites 5′ of SLC26A9. These findings replicated upon re-analysis of scRNA data from 4 independent studies. DNA fragments derived from the 5′ region of SLC26A9 bearing variants from the low risk haplotype generated 12% to 20% higher levels of expression in PANC-1 and CFPAC-1 cells compared to the high risk haplotype (P-values: 2.00E-3 to 5.15E-9). Taken together, our findings indicate that an increase in SLC26A9 expression in ductal cells of the pancreas delays the age-at-onset of diabetes, thereby suggesting a CFTR-agnostic treatment for a major complication of CF.
Anh-Thu N. Lam, Melis A. Aksit, Briana Vecchio-Pagan, Celeste A. Shelton, Derek L. Osorio, Arianna F. Anzmann, Loyal A. Goff, David C. Whitcomb, Scott M. Blackman, Garry R. Cutting
We report on 2 patients with compound heterozygous mutations in forkhead box N1 (FOXN1), a transcription factor essential for thymic epithelial cell (TEC) differentiation. TECs are critical for T cell development. Both patients had a presentation consistent with T–/loB+NK+ SCID, with normal hair and nails, distinct from the classic nude/SCID phenotype in individuals with autosomal-recessive FOXN1 mutations. To understand the basis of this phenotype and the effects of the mutations on FOXN1, we generated mice using CRISPR-Cas9 technology to genocopy mutations in 1 of the patients. The mice with the Foxn1 compound heterozygous mutations had thymic hypoplasia, causing a T–B+NK+ SCID phenotype, whereas the hair and nails of these mice were normal. Characterization of the functional changes due to the Foxn1 mutations revealed a 5–amino acid segment at the end of the DNA-binding domain essential for the development of TECs but not keratinocytes. The transcriptional activity of this Foxn1 mutant was partly retained, indicating a region that specifies TEC functions. Analysis of an additional 9 FOXN1 mutations identified in multiple unrelated patients revealed distinct functional consequences contingent on the impact of the mutation on the DNA-binding and transactivation domains of FOXN1.
Qiumei Du, Larry K. Huynh, Fatma Coskun, Erika Molina, Matthew A. King, Prithvi Raj, Shaheen Khan, Igor Dozmorov, Christine M. Seroogy, Christian A. Wysocki, Grace T. Padron, Tyler R. Yates, M. Louise Markert, M. Teresa de la Morena, Nicolai S.C. van Oers
Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly-inherited diseases—where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function (GoF) allele—is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapeutic for Charcot-Marie-Tooth type 2D (CMT2D), caused by dominant mutations in glycyl tRNA-synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks-of-age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least one year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof-of-concept for gene therapy approaches for dominant neuromuscular diseases.
Kathryn H. Morelli, Laurie B. Griffin, Nettie K. Pyne, Lindsay M. Wallace, Allison M. Fowler, Stephanie N. Oprescu, Ryuichi Takase, Na Wei, Rebecca Meyer-Schuman, Dattatreya Mellacheruvu, Jacob O. Kitzman, Samuel G. Kocen, Timothy J. Hines, Emily L. Spaulding, James R. Lupski, Alexey Nesvizhskii, Pedro Mancias, Ian J. Butler, Xiang-Lei Yang, Ya-Ming Hou, Anthony Antonellis, Scott Q. Harper, Robert W. Burgess
Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDS), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in SSBP1 in five unrelated families, leading to the R38Q and R107Q amino-acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a new revised SSBP1 crystal structure. Structural analysis suggests that both mutations affect dimer interactions and presumably distort the DNA binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication and induces mtDNA depletion. Our study, showing that mutations in SSBP1 cause a novel form of dominant optic atrophy frequently accompanied with foveopathy, brings new insights into mtDNA maintenance disorders.
Camille Piro-Mégy, Emmanuelle Sarzi, Aleix Tarrés-Solé, Marie Péquignot, Fenna Hensen, Mélanie Quilès, Gaël Manes, Arka Chakraborty, Audrey Sénéchal, Béatrice Bocquet, Chantal Cazevieille, Agathe Roubertie, Agnès Müller, Majida Charif, David Goudenège, Guy Lenaers, Helmut Wilhelm, Ulrich Kellner, Nicole Weisschuh, Bernd Wissinger, Xavier Zanlonghi, Christian Hamel, Johannes N. Spelbrink, Maria Solà, Cécile Delettre