Rare diseases are underrepresented in biomedical research, leading to insufficient awareness. Zhu-Tokita-Takenouchi-Kim (ZTTK) syndrome is a rare disease caused by genetic alterations that result in heterozygous loss-of-function of SON. While ZTTK syndrome patients suffer from numerous symptoms, the lack of model organisms hampers our understanding of SON and this complex syndrome. Here, we developed Son haploinsufficiency (Son+/−) mice as a model of ZTTK syndrome and identified the indispensable roles of Son in organ development and hematopoiesis. Son+/− mice recapitulated clinical symptoms of ZTTK syndrome, including growth retardation, cognitive impairment, skeletal abnormalities, and kidney agenesis. Furthermore, we identified hematopoietic abnormalities in Son+/− mice, including leukopenia and immunoglobulin deficiency, similar to those observed in human patients. Surface marker analyses and single-cell transcriptome profiling of hematopoietic stem and progenitor cells revealed that Son haploinsufficiency shifts cell fate more toward the myeloid lineage but compromises lymphoid lineage development by reducing genes required for lymphoid and B-cell lineage specification. Additionally, Son haploinsufficiency causes inappropriate activation of erythroid genes and impaired erythropoiesis. These findings highlight the importance of the full gene expression of Son in multiple organs. Our model serves as an invaluable research tool for this rare disease and related disorders associated with SON dysfunction.
Lana Vukadin, Bohye Park, Mostafa Mohamed, Huashi Li, Amr Elkholy, Alex Torrelli-Diljohn, Jung-Hyun Kim, Kyuho Jeong, James M. Murphy, Caitlin A. Harvey, Sophia Dunlap, Leah Gehrs, Hanna Lee, Hyung-Gyoon Kim, Jay Prakash Sah, Seth N. Lee, Denise Stanford, Robert A. Barrington, Jeremy B. Foote, Anna G. Sorace, Robert S. Welner, Blake E. Hildreth III, Ssang-Taek Steve Lim, Eun-Young Erin Ahn
Pseudohypoparathyroidism type 1B (PHP1B) is caused by aberrant genomic imprinting at the GNAS gene. Defining the underlying genetic cause in new patients is challenging because various genetic alterations (e.g., deletions, insertions) within the GNAS genomic region, including the neighboring STX16 gene, can cause PHP1B, and the genotype-epigenotype correlation has not been clearly established. Here, by analyzing PHP1B patients with a wide variety of genotypes and epigenotypes, we identified a GNAS differentially methylated region (DMR) of distinct diagnostic value. This region, GNAS AS2, was hypomethylated in patients with genetic alterations located centromeric but not telomeric of this DMR. The AS2 methylation status was captured by a single probe of the methylation-sensitive multiplex ligation-dependent probe amplification (MS-MLPA) assay utilized to diagnose PHP1B. In human embryonic stem cells, where NESP55 transcription regulates GNAS methylation status on the maternal allele, AS2 methylation depended on two imprinting control regions (STX16-ICR and NESP-ICR) essential for NESP55 transcription. These results suggest that the AS2 methylation status in PHP1B patients reflects the position at which the genetic alteration affects NESP55 transcription during an early embryonic period. Therefore, AS2 methylation levels enable mechanistic PHP1B categorization based on genotype-epigenotype correlation and thus help identify the underlying molecular defect in patients.
Yorihiro Iwasaki, Monica Reyes, Harald Jüppner, Murat Bastepe
A distinct adipose tissue distribution pattern was observed in patients with methylmalonyl-CoA mutase deficiency, an inborn error of branched-chain amino acid (BCAA) metabolism, characterized by centripetal obesity with proximal upper and lower extremities fat deposition and paucity of visceral fat, that resembles familial multiple lipomatosis syndrome. To explore brown and white fat physiology in methylmalonic acidemia (MMA), body composition, adipokines and inflammatory markers were assessed in 46 MMA subjects and 99 matched controls. Fibroblast growth factor-21 (FGF21) levels were associated with acyl-coenzyme A accretion, aberrant methylmalonylation in adipose tissue, and an attenuated inflammatory cytokine profile. In parallel, brown and white fat were examined in a liver-specific transgenic MMA mouse model (Mmut-/-;TgINS-Alb-Mmut). The MMA mice exhibited abnormal non-shivering thermogenesis with whitened brown fat and had an ineffective transcriptional response to cold stress. Treatment of the MMA mice with bezafibrates led to clinical improvement with beiging of subcutaneous fat depots, which resembled the distribution seen in the patients. These studies defined what we believe to be a novel lipodystrophy phenotype in patients with defects in the terminal steps of BCAA oxidation and demonstrated that beiging of subcutaneous adipose tissue in MMA could readily be induced with small molecules.
Irini Manoli, Justin R. Sysol, PamelaSara E. Head, Madeline W. Epping, Oksana Gavrilova, Melissa K. Crocker, Jennifer L. Sloan, Stefanos A. Koutsoukos, Cindy X. Wang, Yiouli P. Ktena, Sophia Mendelson, Alexandra R. Pass, Patricia M. Zerfas, Victoria J. Hoffmann, Hilary J. Vernon, Laura A. Fletcher, James C. Reynolds, Maria G. Tsokos, Constantine A. Stratakis, Stephan D. Voss, Kong Y. Chen, Rebecca J. Brown, Ada Hamosh, Gerard T. Berry, Xiaoyuan Chen, Jack A. Yanovski, Charles P. Venditti
Genetic modifications leading to pain insensitivity phenotypes are rare but can provide invaluable insights into the molecular biology of pain and reveal novel targets for analgesic drugs. Pain insensitivity typically results from Mendelian loss-of-function mutations in genes expressed in nociceptive (pain-sensing) dorsal root ganglion (DRG) neurons that connect the body to the spinal cord. We document a novel pain insensitivity mechanism arising from gene overexpression in individuals with the rare 7q11.23 duplication syndrome (Dup7), who have three copies of the approximately 1.5 megabase Williams syndrome (WS) critical region. Based on parental accounts and pain ratings, people with Dup7, mainly children in this study, are pain insensitive following serious injury to skin, bones, teeth, or viscera. In contrast, diploid siblings (two copies) and people with WS (one copy) show standard reactions to painful events. A converging series of human assessments and cross-species cell biological and transcriptomic studies identified one likely candidate in the WS critical region, STX1A, as underlying the pain insensitivity phenotype. STX1A codes for the synaptic vesicle fusion protein Syntaxin1A and neuropeptide release studies from nociceptive DRG neurons, show that excess syntaxin1A compromises exocytosis which when extrapolated to Dup7 individuals, produces a “genetic analgesia” and new potential routes to pain control.
Michael J. Iadarola, Matthew R. Sapio, Amelia J. Loydpierson, Carolyn B. Mervis, Jill C. Fehrenbacher, Michael R. Vasko, Dragan Maric, Daniel P. Eisenberg, Tiffany A. Nash, J. Shane Kippenhan, Madeline H. Garvey, Andrew J. Mannes, Michael D. Gregory, Karen F. Berman
The clinical spectrum of thyrotropin receptor (TSHR)-mediated diseases varies from loss-of-function mutations causing congenital hypothyroidism to constitutively active mutations (CAMs) leading to nonautoimmune hyperthyroidism (NAH). Variation at the TSHR locus has also been associated with altered lipid and bone metabolism and autoimmune thyroid diseases. However, the extrathyroidal roles of TSHR, and the mechanisms underlying phenotypic variability among TSHR-mediated diseases remain unclear. Here we identified and characterized TSHR variants and factors involved in phenotypic variability in different patient cohorts, the FinnGen database, and a mouse model. TSHR CAMs were found in all 16 patients with NAH, with one CAM in an unexpected location in the extracellular leucine-rich repeat domain (p.S237N) and another in the transmembrane domain (p.I640V) in two families with distinct hyperthyroid phenotypes. In addition, screening of the FinnGen database revealed rare functional variants, as well as distinct common non-coding TSHR SNPs significantly associated with thyroid phenotypes, but no other significant association between TSHR variants and over 2,000 non-thyroid disease endpoints. Finally, our TSHR M453T knock-in model revealed that the phenotype was dependent on the mutation´s signaling properties and was ameliorated by increased iodine intake. In summary, our data shows that TSHR-mediated disease risk can be modified by variants at the TSHR locus both inside and outside the coding region, and by altered TSHR-signaling and dietary iodine, supporting the need for personalized treatment strategies.
Kristiina Makkonen, Meeri Jännäri, Luís Crisóstomo, Matilda Kuusi, Konrad Patyra, Vladyslav Melnyk, Veli M. Linnossuo, Johanna O. Ojala, Rowmika Ravi, Christoffer Löf, Juho-Antti Mäkelä, Päivi J. Miettinen, Saila Laakso, Marja Ojaniemi, Jarmo Jääskeläinen, Markku Laakso, Filip Bossowski, Beata Sawicka, Karolina Stożek, Artur Bossowski, Gunnar Kleinau, Patrick Scheerer, Finngen Finngen, Mary Pat Reeve, Jukka Kero
Cutaneous neurofibromas (cNFs) are benign Schwann cell (SC) tumors arising from subepidermal glia. Neurofibromatosis Type 1 (NF1) individuals may develop thousands of cNFs, greatly affecting their quality of life. cNF growth is driven by the proliferation of NF1(-/-) SCs and their interaction with NF1(+/-) microenvironment. We analyzed the crosstalk between human cNF-derived SCs and fibroblasts (FBs), identifying an expression signature specific to SC-FB interaction. We validated the secretion of proteins involved in immune cell migration, suggesting a role of SC-FB crosstalk in immune cell recruitment. The signature also captured components of developmental signaling pathways, including the cAMP-elevator G protein-coupled receptor 68 (GPR68). Activation of Gpr68 by Ogerin in combination with the MEKi Selumetinib reduced viability and induced differentiation and death of human cNF-derived primary SCs, a result corroborated using an iPSC-derived 3D neurofibromasphere model. Similar results were obtained using other Gpr68 activators or cAMP analogs/adenylyl cyclase activators in combination with Selumetinib. Interestingly, whereas primary SC cultures reset proliferation after removal of single Selumetib treatment, Ogerin-Selumetinib combo elicited a permanent halt on SC expansion, also after drug removal. These results indicate that unbalancing the Ras and cAMP pathways by combining MEKi and cAMP elevators arises as a potential treatment for cNFs.
Helena Mazuelas, Miriam Magallón-Lorenz, Itziar Uriarte-Arrazola, Alejandro Negro, Inma Rosas, Ignacio Blanco, Elisabeth Castellanos, Conxi Lázaro, Bernat Gel, Meritxell Carrió, Eduard Serra
Dissemination within the peritoneal cavity is a main determinant of poor patient outcomes from high-grade serous carcinomas (HGSCs). The dissemination process is poorly understood from a cancer evolutionary perspective. We reconstructed the evolutionary trajectories across a median of five tumor sites and regions from each of 23 patients (n=108 samples) based on deep whole-exome sequencing. Polyclonal cancer origin was detected in one patient. Ovarian tumors had more complex subclonal architectures than other intra-peritoneal tumors in each patient, which indicated that tumors developed earlier in the ovaries. Three common modes of dissemination were identified, including monoclonal (27%) or polyclonal dissemination of monophyletic (linear; 50%) or polyphyletic (branched; 23%) subclones. Mutation profiles of initial or disseminated clones varied greatly among cancers, but recurrent mutations were found in seven cancer-critical genes, such as TP53, BRCA1, BRCA2, DNMT3A, and in the PI3K/AKT1 pathway. Disseminated clones developed late in the evolutionary trajectory models of most cancers, in particular in cancers with DNA damage repair deficiency. Polyclonal dissemination was predicted to occur predominantly as a single and rapid wave, but chemotherapy exposure was associated with higher genomic diversity of disseminated clones. In conclusion, we described three common evolutionary dissemination modes across HGSCs and proposed factors associated with dissemination diversity.
Anita Sveen, Bjarne Johannessen, Solveig M.K. Klokkerud, Sigrid M. Kraggerud, Leonardo A. Meza-Zepeda, Merete Bjørnslett, Katharina Bischof, Ola Myklebost, Kjetil Taskén, Rolf I. Skotheim, Anne Dørum, Ben Davidson, Ragnhild A. Lothe
Long noncoding RNAs (lncRNAs) regulate the expression of protein-coding genes and have been shown to play important roles in inflammatory skin diseases. However, we still have limited understanding of the functional impact of lncRNAs in skin, partly due to their tissue specificity and lower expression levels compared with protein-coding genes. We compiled a comprehensive list of 18,517 lncRNAs from different sources and studied their expression profiles in 834 RNA-Seq samples from multiple inflammatory skin conditions and cytokine-stimulated keratinocytes. Applying a balanced random forest to predict involvement in biological functions, we achieved a median AUROC of 0.79 in 10-fold cross-validation, identifying significant DNA binding domains (DBDs) for 39 lncRNAs. G18244, a skin-expressing lncRNA predicted for IL-4/IL-13 signaling in keratinocytes, was highly correlated in expression with F13A1, a protein-coding gene involved in macrophage regulation, and we further identified a significant DBD in F13A1 for G18244. Reflecting clinical implications, AC090198.1 (predicted for IL-17 pathway) and AC005332.6 (predicted for IFN-γ pathway) had significant negative correlation with the SCORAD metric for atopic dermatitis. We also utilized single-cell RNA and spatial sequencing data to validate cell type specificity. Our research demonstrates lncRNAs have important immunological roles and can help prioritize their impact on inflammatory skin diseases.
Matthew T. Patrick, Sutharzan Sreeskandarajan, Alanna Shefler, Rachael Wasikowski, Mrinal K. Sarkar, Jiahan Chen, Tingting Qin, Allison C. Billi, J. Michelle Kahlenberg, Errol Prens, Alain Hovnanian, Stephan Weidinger, James T. Elder, Chao-Chung Kuo, Johann E. Gudjonsson, Lam C. Tsoi
Weaver syndrome is a Mendelian disorder of the epigenetic machinery (MDEM) caused by germline pathogenic variants in EZH2, which encodes the predominant H3K27 methyltransferase and key enzymatic component of Polycomb repressive complex 2 (PRC2). Weaver syndrome is characterized by striking overgrowth and advanced bone age, intellectual disability, and distinctive facies. We generated a mouse model for the most common Weaver syndrome missense variant, EZH2 p.R684C. Ezh2R684C/R684C mouse embryonic fibroblasts (MEFs) showed global depletion of H3K27me3. Ezh2R684C/+ mice had abnormal bone parameters indicative of skeletal overgrowth, and Ezh2R684C/+ osteoblasts showed increased osteogenic activity. RNA-seq comparing osteoblasts differentiated from Ezh2R684C/+ and Ezh2+/+ bone marrow mesenchymal stem cells (BM-MSCs) indicated collective dysregulation of the BMP pathway and osteoblast differentiation. Inhibition of the opposing H3K27 demethylases KDM6A/6B substantially reversed the excessive osteogenesis in Ezh2R684C/+ cells both at the transcriptional and phenotypic levels. This supports both the ideas that writers and erasers of histone marks exist in a fine balance to maintain epigenome state, and that epigenetic modulating agents have therapeutic potential for the treatment of MDEMs.
Christine W. Gao, WanYing Lin, Ryan C. Riddle, Priyanka Kushwaha, Leandros Boukas, Hans T. Björnsson, Kasper D. Hansen, Jill A. Fahrner
HNF1A haploinsufficiency underlies the most common form of human monogenic diabetes (HNF1A-MODY) and hypomorphic HNF1A variants confer type 2 diabetes risk, but a lack of experimental systems for interrogating mature human islets has limited our understanding of how the transcription factor HNF1α regulates adult islet function. Here, we combined conditional genetic targeting in human islet cells, RNA sequencing, chromatin mapping with Cleavage Under Targets & Release Using Nuclease (CUT&RUN), and transplantation-based assays to determine HNF1α-regulated mechanisms in adult human pancreatic α and β cells. Short hairpin RNA-mediated (shRNA) suppression of HNF1A in primary human pseudoislets led to blunted insulin output and dysregulated glucagon secretion after transplantation in mice, recapitulating phenotypes observed in diabetic patients. These deficits corresponded with altered expression of genes encoding factors critical for hormone secretion, including calcium channel subunits, ATPase transporters and extracellular matrix constituents. Additionally, HNF1A loss led to upregulation of transcriptional repressors, providing evidence for a mechanism of transcriptional de-repression through HNF1α. CUT&RUN mapping of HNF1α DNA-binding sites in primary human islets imputed a subset of HNF1α-regulated genes as direct targets. These data elucidate mechanistic links between HNF1A loss and diabetic phenotypes in mature human α and β cells.
Mollie F. Qian, Romina J. Bevacqua, Vy M.N. Coykendall, Xiong Liu, Weichen Zhao, Charles A. Chang, Xueying Gu, Xiao-Qing Dai, Patrick E. MacDonald, Seung K. Kim
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