Sulfate plays a pivotal role in numerous physiological processes in the human body, including bone and cartilage health. A role of the anion transporter SLC26A1 (Sat1) for sulfate reabsorption in the kidney is supported by the observation of hyposulfatemia and hypersulfaturia in Slc26a1-knockout mice. The impact of SLC26A1 on sulfate homeostasis in humans remains to be defined. By combining clinical genetics, functional expression assays, and population exome analysis, we identify SLC26A1 as a sulfate transporter in humans and experimentally validate several loss-of-function alleles. Whole-exome sequencing from a patient presenting with painful perichondritis, hyposulfatemia, and renal sulfate wasting revealed a homozygous mutation in SLC26A1, which has not been previously described to the best of our knowledge. Whole-exome data analysis of more than 5,000 individuals confirmed that rare, putatively damaging SCL26A1 variants were significantly associated with lower plasma sulfate at the population level. Functional expression assays confirmed a substantial reduction in sulfate transport for the SLC26A1 mutation of our patient, which we consider to be novel, as well as for the additional variants detected in the population study. In conclusion, combined evidence from 3 complementary approaches supports SLC26A1 activity as a major determinant of sulfate homeostasis in humans. In view of recent evidence linking sulfate homeostasis with back pain and intervertebral disc disorder, our study identifies SLC26A1 as a potential target for modulation of musculoskeletal health.
Anja Pfau, Karen I. López-Cayuqueo, Nora Scherer, Matthias Wuttke, Annekatrin Wernstedt, Daniela González Fassrainer, Desiree E.C. Smith, Jiddeke M. van de Kamp, Katharina Ziegeler, Kai-Uwe Eckardt, Friedrich C. Luft, Peter S. Aronson, Anna Köttgen, Thomas J. Jentsch, Felix Knauf
Preimplantation embryo arrest (PREMBA) is a common cause of female infertility and recurrent failure of assisted reproductive technology. However, the genetic basis of PREMBA is largely unrevealed. Here, using whole-exome sequencing data from 606 women experiencing PREMBA compared with 2,813 controls, we performed a population and gene–based burden test and identified a candidate gene, karyopherin subunit α7 (KPNA7). In vitro studies showed that identified sequence variants reduced KPNA7 protein levels, impaired KPNA7 capacity for binding to its substrate ribosomal L1 domain-containing protein 1 (RSL1D1), and affected KPNA7 nuclear transport activity. Comparison between humans and mice suggested that mouse KPNA2, rather than mouse KPNA7, acts as an essential karyopherin in embryonic development. Kpna2–/– female mice showed embryo arrest due to zygotic genome activation defects, recapitulating the phenotype of human PREMBA. In addition, female mice with an oocyte-specific knockout of Rsl1d1 recapitulated the phenotype of Kpna2–/– mice, demonstrating the vital role of substrate RSL1D1. Finally, complementary RNA (cRNA) microinjection of human KPNA7, but not mouse Kpna7, was able to rescue the embryo arrest phenotype in Kpna2–/– mice, suggesting mouse KPNA2 might be a homologue of human KPNA7. Our findings uncovered a mechanistic understanding for the pathogenesis of PREMBA, which acts by impairing nuclear protein transport, and provide a diagnostic marker for PREMBA patients.
Wenjing Wang, Yoichi Miyamoto, Biaobang Chen, Juanzi Shi, Feiyang Diao, Wei Zheng, Qun Li, Lan Yu, Lin Li, Yao Xu, Ling Wu, Xiaoyan Mao, Jing Fu, Bin Li, Zheng Yan, Rong Shi, Xia Xue, Jian Mu, Zhihua Zhang, Tianyu Wu, Lin Zhao, Weijie Wang, Zhou Zhou, Jie Dong, Qiaoli Li, Li Jin, Lin He, Xiaoxi Sun, Ge Lin, Yanping Kuang, Lei Wang, Qing Sang
Mutations of G protein coupled receptors (GPCRs) cause various human diseases, but the mechanistic details are limited. Here we establish p.E303K in the gene encoding the endothelin receptor type A (ETAR/EDNRA) as a recurrent mutation causing Mandibulofacial dysostosis with alopecia (MFDA), with craniofacial changes similar to those caused by p.Y129F. Mouse models carrying either of these missense mutations exhibit a partial maxillary-to-mandibular transformation, which is rescued by deleting the ligand endothelin 3 (ET3/EDN3). Pharmacological experiments confirmed the causative ETAR mutations as gain-of-function, dependent on ET3. To elucidate how an amino acid substitution far from the ligand binding site can increase ligand affinity, we used molecular dynamics (MD) simulations. E303 is located at the intracellular end of transmembrane domain 6, and its replacement by a lysine increases flexibility of this portion of the helix, thus favoring G-protein binding and leading to G-protein-mediated enhancement of agonist affinity. The Y129F mutation located under the ligand binding pocket reduces the sodium-water network, thereby affecting the extracellular portion of helices in favor of ET3 binding. These findings provide insight into the pathogenesis of MFDA and into allosteric mechanisms regulating GPCR function, that may provide the basis for drug design targeting GPCRs.
Yukiko Kurihara, Toru Ekimoto, Christopher T. Gordon, Yasunobu Uchijima, Ryo Sugiyama, Taro Kitazawa, Akiyasu Iwase, Risa Kotani, Rieko Asai, Véronique Pingault, Mitsunori Ikeguchi, Jeanne Amiel, Hiroki Kurihara
Multiple genetic loci have been reported for progeroid syndromes. However, the molecular defects in some extremely rare forms of progeria have yet to be elucidated. Here we report a 21-year-old man of Chinese origin who had a novel autosomal recessive form of progeria, characterized by severe dwarfism, mandibular hypoplasia, hyperopia and partial lipodystrophy. Analyses of exome sequencing data of the entire family revealed only one rare homozygous missense variant, (c.86C>T; p.Pro29Leu), in TOMM7 in the proband, while the parents and two unaffected siblings were heterozygous for the variant. TOMM7, a nuclear gene, encodes a translocase in the outer mitochondrial membrane. The TOMM complex constitutes the outer membrane pore for import of several preproteins into mitochondria. Proteomics analyses of mitochondria from cultured fibroblasts of the proband, as compared to control fibroblasts, revealed increases in several proteins involved in oxidative phosphorylation, but reduced abundance of proteins involved in the phospholipid metabolism. We also observed elevated basal and maximal oxygen consumption rates in the fibroblasts from the proband as compared to control fibroblasts. We conclude that altered mitochondrial protein import due to loss of function bi-allelic variant in TOMM7 can cause severe growth retardation and progeroid features.
Abhimanyu Garg, Wee-Teik Keng, Zhenkang Chen, Adwait Amod Sathe, Chao Xing, Pavithira Devi Kailasam, Yanqiu Shao, Nicholas P. Lesner, Claire B. Llamas, Anil K. Agarwal, Prashant Mishra
22q11.2 deletion syndrome (22q11.2DS) is the most common human chromosomal microdeletion, causing developmentally linked congenital malformations; thymus hypoplasia, hypoparathyroidism and/or cardiac defects. Thymus hypoplasia leads to T cell lymphopenia, which most often results in mild SCID. Despite decades of research, the molecular underpinnings leading to thymus hypoplasia in 22q11.2DS remain unknown. Comparing embryonic thymuses from mouse models of 22q11.2DS (Tbx1neo2/neo2) revealed similar proportions of mesenchymal-, epithelial- and hematopoietic- cell types as controls. Yet, the small thymuses were growth restricted in fetal organ cultures. Replacement of Tbx1neo2/neo2 thymus mesenchymal cells with normal ones restored tissue growth. Comparative single cell RNA sequencing of embryonic thymuses uncovered 17 distinct cell subsets, with transcriptome differences predominant in the 5 mesenchymal subsets from the Tbx1neo2/neo2 line. Transcripts impacted include extracellular matrix (ECM) proteins, consistent with increased collagen deposition seen in the small thymuses. Attenuating collagen cross-links with minoxidil restored thymus tissue expansion for hypoplastic lobes. In colony forming assays, the Tbx1neo2/neo2-derived mesenchymal cells had reduced expansion potential, contrasting the normal growth of thymus epithelial cells. These findings suggest that mesenchymal cells are causal to the small embryonic thymuses in 22q11.2DS mouse models, correctable by substituting with normal mesenchyme.
Pratibha Bhalla, Qiumei Du, Ashwani Kumar, Chao Xing, Angela Moses, Igor Dozmorov, Christian A. Wysocki, Ondine B. Cleaver, Timothy J. Pirolli, Mary Louise Markert, M. Teresa de la Morena, Antonio Baldini, Nicolai S.C. van Oers
Initiation and maintenance of transcriptional states are critical for controlling normal tissue homeostasis and differentiation. Cyclin Dependent Kinases CDK8/CDK19 (Mediator kinase) are regulatory components of Mediator, a highly conserved complex that orchestrates enhancer-mediated transcriptional output. While Mediator kinase has been implicated in the transcription of genes necessary for development and growth, its function in mammals has not been well defined. Using a suite of genetically defined models and pharmacological inhibitors, we show that Cdk8/19 function in a redundant manner to regulate intestinal lineage-specification in human and mouse. Mechanistically, we find that the Mediator kinase module binds and phosphorylates key components of the chromatin remodelling complex SWI/SNF in intestinal epithelial cells. Concomitantly, SWI/SNF and MED12-Mediator co-localise at distinct lineage-specifying enhancers in a CDK8/19 dependent manner. As such, these studies reveal a novel transcriptional mechanism of intestinal cell specification, coordinated by the interaction between the chromatin remodelling complex SWI/SNF and Mediator kinase.
Marius V. Dannappel, Danxi Zhu, Xin Sun, Hui Kheng Chua, Marle Poppelaars, Monica Suehiro, Subash Khadka, Terry C.C. Lim Kam Sian, Dhanya Sooraj, Melissa Loi, Hugh Gao, Daniel Croagh, Roger J. Daly, Pouya Faridi, Thomas Boyer, Ron Firestein
Autism spectrum disorder (ASD) represents a group of neurodevelopmental phenotypes with a strong genetic component. Excess of likely gene-disruptive (LGD) mutations of GIGYF1 was implicated in ASD. Here, we reported that GIGYF1 was the second most mutated gene among known ASD high-confidence risk genes. We investigated the inheritance of 46 GIGYF1 LGD variants, including the highly recurrent mutation, c.333del:p.L111Rfs*234. Inherited GIGYF1 heterozygous LGD variants were 1.8 times more common than de novo mutations. Unlike most high-confidence genes, ASD individuals with GIGYF1 LGD variants were less likely to have cognitive impairments. Using a Gigyf1 conditional knockout mouse model, we showed that haploinsufficiency in the developing brain led to social impairments without significant cognitive impairments. In contrast, homozygous mice showed more severe social disability as well as cognitive impairments. Gigyf1 deficiency in mice led to a reduction of upper layer cortical neurons accompanied by decreased proliferation and increased differentiation of neural progenitor cells. We showed that GIGYF1 regulated the recycling of IGF-1R to cell surface. Knockout of GIGYF1 led to a decreased level of IGF-1R on the cell surface disrupting the IGF-1R/ERK signaling pathway. In summary, our findings showed that GIGYF1 was a regulator of IGF-1R recycling. Haploinsufficiency of GIGYF1 was associated with autistic behaviors likely through interference with IGR-1R/ERK signaling pathway.
Guodong Chen, Bin Yu, Senwei Tan, Jieqiong Tan, Xiangbin Jia, Qiumeng Zhang, Xiaolei Zhang, Qian Jiang, Yue Hua, Yaoling Han, Shengjie Luo, Kendra Hoekzema, Raphael A. Bernier, Rachel K. Earl, Evangeline C. Kurtz-Nelson, Michaela J. Idleburg, Suneeta Madan Khetarpal, Rebecca Clark, Jessica Sebastian, Alberto Fernandez-Jaen, Sara Alvarez, Staci D. King, Luiza L.P. Ramos, Mara Lucia S.F. Santos, Donna M. Martin, Dan Brooks, Joseph D. Symonds, Ioana Cutcutache, Qian Pan, Zhengmao Hu, Ling Yuan, Evan E. Eichler, Kun Xia, Hui Guo
Mitochondrial stress triggers a response in the cell’s mitochondria and nucleus, but how these stress responses are coordinated in vivo is poorly understood. Here, we characterize a family with myopathy caused by a dominant p.G58R mutation in the mitochondrial protein CHCHD10. To understand the disease etiology, we developed a knock-in mouse model and found that mutant CHCHD10 aggregates in affected tissues, applying a toxic protein stress to the inner mitochondrial membrane. Unexpectedly, survival of CHCHD10 knock-in mice depended on a protective stress response mediated by OMA1. The OMA1 stress response acted both locally within mitochondria, causing mitochondrial fragmentation, and signaled outside the mitochondria, activating the integrated stress response through cleavage of DELE1. We additionally identified an isoform switch in the terminal complex of the electron transport chain as a component of this response. Our results demonstrate that OMA1 is critical for neonatal survival conditionally in the setting of inner mitochondrial membrane stress, coordinating local and global stress responses to reshape the mitochondrial network and proteome.
Mario K. Shammas, Xiaoping Huang, Beverly P. Wu, Evelyn Fessler, Insung Song, Nicholas P. Randolph, Yan Li, Christopher K.E. Bleck, Danielle A. Springer, Carl Fratter, Ines A. Barbosa, Andrew F. Powers, Pedro M. Quirós, Carlos Lopez-Otin, Lucas T. Jae, Joanna Poulton, Derek P. Narendra
Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders due to impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report five probands from four families who presented with ptosis and ophthalmoplegia, plus other manifestations and multiple mtDNA deletions in muscle. We identified three RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and two homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.
Jonathan Shintaku, Wolfgang M. Pernice, Wafaa Eyaid, Jeevan B. GC, Zuben P. Brown, Marti Juanola-Falgarona, Javier Torres-Torronteras, Ewen W. Sommerville, Debby M.E.I. Hellebrekers, Emma L. Blakely, Alan Donaldson, Ingrid M.B.H. van de Laar, Cheng-Shiun Leu, Ramon Marti, Joachim Frank, Kurenai Tanji, David A. Koolen, Richard J. Rodenburg, Patrick F. Chinnery, H.J.M. Smeets, Gráinne S. Gorman, Penelope E. Bonnen, Robert W. Taylor, Michio Hirano
Many neurodegenerative disorders are caused by abnormal accumulation of misfolded proteins. In spinocerebellar ataxia type 1 (SCA1), accumulation of polyglutamine-expanded (polyQ-expanded) ataxin-1 (ATXN1) causes neuronal toxicity. Lowering total ATXN1, especially the polyQ-expanded form, alleviates disease phenotypes in mice, but the molecular mechanism by which the mutant ATXN1 is specifically modulated is not understood. Here, we identified 22 mutant ATXN1 regulators by performing a cross-species screen of 7787 and 2144 genes in human cells and Drosophila eyes, respectively. Among them, transglutaminase 5 (TG5) preferentially regulated mutant ATXN1 over the WT protein. TG enzymes catalyzed cross-linking of ATXN1 in a polyQ-length–dependent manner, thereby preferentially modulating mutant ATXN1 stability and oligomerization. Perturbing Tg in Drosophila SCA1 models modulated mutant ATXN1 toxicity. Moreover, TG5 was enriched in the nuclei of SCA1-affected neurons and colocalized with nuclear ATXN1 inclusions in brain tissue from patients with SCA1. Our work provides a molecular insight into SCA1 pathogenesis and an opportunity for allele-specific targeting for neurodegenerative disorders.
Won-Seok Lee, Ismael Al-Ramahi, Hyun-Hwan Jeong, Youjin Jang, Tao Lin, Carolyn J. Adamski, Laura A. Lavery, Smruti Rath, Ronald Richman, Vitaliy V. Bondar, Elizabeth Alcala, Jean-Pierre Revelli, Harry T. Orr, Zhandong Liu, Juan Botas, Huda Y. Zoghbi