Genetics
Abstract
ZIP8 is a metal transporter with a role in manganese (Mn) homeostasis. A common genetic variant in ZIP8 (rs13107325; A391T) ranks in the top 10 of pleiotropic single nucleotide polymorphisms (SNP) identified in genome-wide association studies, including associations with an increased risk of schizophrenia, obesity, Crohn’s disease, and reduced blood Mn. Here, we used CRISPR/Cas9-mediated knock-in (KI) to generate a mouse model of ZIP8 A391T (mouse Zip8 393T-KI). Recapitulating the SNP association with blood Mn, blood Mn is reduced in Zip8 393T-KI mice. There is restricted abnormal tissue Mn homeostasis with decreases in liver and kidney Mn and reciprocal increase in biliary Mn to provide in vivo evidence of hypomorphic Zip8 function. Upon challenge in a chemical-induced colitis model, male Zip8 393T-KI mice exhibited enhanced disease susceptibility. ZIP8 391-Thr associated with reduced triantennary plasma N-glycan species in a population-based cohort to define a genotype-specific glycophenotype hypothesized to be linked to Mn-dependent glycosyltransferase activity. This glycophenotype was maintained in a cohort of Crohn’s disease patients. These data and the pleiotropic disease associations with ZIP8 391-Thr suggest underappreciated roles of Mn homeostasis in compex human disease.
Authors
Laxmi Sunuwar, Azra Frkatovic, Sodbo Sharapov, Qinchuan Wang, Heather Neu, Xinqun Wu, Talin Haritunians, Fengyi Wan, Sarah L. J. Michel, Shaoguang Wu, Dermot McGovern, Gordan Lauc, Mark Donowitz, Cynthia L. Sears, Joanna M.P. Melia
Abstract
More than 90% of autoimmune-associated variants are located in noncoding regions, leading to challenges in deciphering the underlying causal roles of functional variants and genes and biological mechanisms. Therefore, to reduce the gap between traditional genetic findings and mechanistic understanding of disease etiologies and clinical drug development, it is important to translate systematically the regulatory mechanisms underlying noncoding variants. Here, we prioritized functional noncoding SNPs with regulatory gene targets associated with 19 autoimmune diseases by incorporating hundreds of immune cell–specific multiomics data. The prioritized SNPs are associated with transcription factor (TF) binding, histone modification, or chromatin accessibility, indicating their allele-specific regulatory roles. Their target genes are significantly enriched in immunologically related pathways and other known immunologically related functions. We found that 90.1% of target genes are regulated by distal SNPs involving several TFs (e.g., the DNA-binding protein CCCTC-binding factor [CTCF]), suggesting the importance of long-range chromatin interaction in autoimmune diseases. Moreover, we predicted potential drug targets for autoimmune diseases, including 2 genes (NFKB1 and SH2B3) with known drug indications on other diseases, highlighting their potential drug repurposing opportunities. Taken together, these findings may provide useful information for future experimental follow-up and drug applications on autoimmune diseases.
Authors
Xiao-Feng Chen, Ming-Rui Guo, Yuan-Yuan Duan, Feng Jiang, Hao Wu, Shan-Shan Dong, Xiao-Rong Zhou, Hlaing Nwe Thynn, Cong-Cong Liu, Lin Zhang, Yan Guo, Tie-Lin Yang
Abstract
Lynch syndrome is the most common colorectal cancer (CRC) hereditary form and it is characterized by DNA mismatch repair (MMR) deficiency. The term Lynch-like syndrome (LLS) is used for patients with MMR-deficient tumors and neither germline mutation in MLH1, MSH2, MSH6, PMS2, or EPCAM, nor MLH1 somatic methylation. Biallelic somatic inactivation or cryptic germline MMR variants undetected during genetic testing have been proposed to be involved. Sixteen patients with early-onset LLS CRC were selected for germline and tumor whole-exome sequencing. Two potentially pathogenic germline MCM8 variants were detected in a LLS male patient with fertility problems. A knockout cellular model for MCM8 was generated by CRISPR-Cas9 and detected genetic variants were produced by mutagenesis. DNA damage, microsatellite instability and mutational signatures were monitored. DNA damage was evident for MCM8KO cells and the analyzed genetic variants. Microsatellite instability and mutational signatures in MCM8KO cells were compatible with the involvement of MCM8 in MMR. Replication in an independent familial cancer cohort detected additional carriers. Unexplained MMR-deficient CRC cases, even showing somatic biallelic MMR inactivation, may be caused by underlying germline defects in genes different than the MMR genes. We suggest MCM8 as a new gene involved in CRC germline predisposition with a recessive pattern of inheritance.
Authors
Mariano Golubicki, Laia Bonjoch, José G. Acuña-Ochoa, Marcos Díaz-Gay, Jenifer Muñoz, Miriam Cuatrecasas, Teresa Ocaña, Soledad Iseas, Guillermo Mendez, Daniel Cisterna, Stephanie A. Schubert, Maartje Nielsen, Tom van Wezel, Yael Goldberg, Eli Pikarsky, Juan Robbio, Enrique Roca, Antoni Castells, Francesc Balaguer, Marina Antelo, Sergi Castellví-Bel
Abstract
Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948–1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn’s function in tubular system organization and normal cardiac function.
Authors
Canzhao Liu, Simone Spinozzi, Wei Feng, Ze’e Chen, Lunfeng Zhang, Siting Zhu, Tongbin Wu, Xi Fang, Kunfu Ouyang, Sylvia M. Evans, Ju Chen
Abstract
Cancer is instigated by mutator phenotypes, including deficient mismatch repair and p53-associated chromosomal instability. More recently, a distinct class of cancers was identified with unusually high mutational loads due to heterozygous amino acid substitutions (most commonly P286R) in the proofreading domain of DNA polymerase ε, the leading strand replicase encoded by POLE. Immunotherapy has revolutionized cancer treatment, but new model systems are needed to recapitulate high mutational burdens characterizing human cancers and permit study of mechanisms underlying clinical responses. Here, we show that activation of a conditional LSL-PoleP286R allele in endometrium is sufficient to elicit in all animals endometrial cancers closely resembling their human counterparts, including very high mutational burden. Diverse investigations uncovered potentially novel aspects of Pole-driven tumorigenesis, including secondary p53 mutations associated with tetraploidy, and cooperation with defective mismatch repair through inactivation of Msh2. Most significantly, there were robust antitumor immune responses with increased T cell infiltrates, accelerated tumor growth following T cell depletion, and unfailing clinical regression following immune checkpoint therapy. This model predicts that human POLE-driven cancers will prove consistently responsive to immune checkpoint blockade. Furthermore, this is a robust and efficient approach to recapitulate in mice the high mutational burdens and immune responses characterizing human cancers.
Authors
Hao-Dong Li, Changzheng Lu, He Zhang, Qing Hu, Junqiu Zhang, Ileana C. Cuevas, Subhransu S. Sahoo, Mitzi Aguilar, Elizabeth G. Maurais, Shanrong Zhang, Xiaojing Wang, Esra A. Akbay, Guo-Min Li, Bo Li, Prasad Koduru, Peter Ly, Yang-Xin Fu, Diego H. Castrillon
Abstract
The α-1-antitrypsin (or alpha-1-antitrypsin, A1AT) Z variant is the primary cause of severe A1AT deficiency and forms polymeric chains that aggregate in the endoplasmic reticulum of hepatocytes. Around 2%–5% of Europeans are heterozygous for the Z and WT M allele, and there is evidence of increased risk of liver disease when compared with MM A1AT individuals. We have shown that Z and M A1AT can copolymerize in cell models, but there has been no direct observation of heteropolymer formation in vivo. To this end, we developed a monoclonal antibody (mAb2H2) that specifically binds to M in preference to Z A1AT, localized its epitope using crystallography to a region perturbed by the Z (Glu342Lys) substitution, and used Fab fragments to label polymers isolated from an MZ heterozygote liver explant. Glu342 is critical to the affinity of mAb2H2, since it also recognized the mild S-deficiency variant (Glu264Val) present in circulating polymers from SZ heterozygotes. Negative-stain electron microscopy of the Fab2H2-labeled liver polymers revealed that M comprises around 6% of the polymer subunits in the MZ liver sample. These data demonstrate that Z A1AT can form heteropolymers with polymerization-inert variants in vivo with implications for liver disease in heterozygous individuals.
Authors
Mattia Laffranchi, Emma L.K. Elliston, Elena Miranda, Juan Perez, Riccardo Ronzoni, Alistair M. Jagger, Nina Heyer-Chauhan, Mark L. Brantly, Annamaria Fra, David A. Lomas, James A. Irving
Abstract
We identified a novel homozygous duplication involving the promoter region and exons 1-4 of RYR2 that is responsible for highly penetrant, exertion-related sudden deaths/cardiac arrests in the Amish community without an overt phenotype to suggest RYR2-mediated catecholaminergic polymorphic ventricular tachycardia (CPVT). Homozygous RYR2-duplication (RYR2-DUP) induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) were generated from two unrelated patients. There was no difference in baseline Ca2+ handling measurements between WT- and the RYR2-DUP-iPSC-CMs lines. However, compared to WT-iPSC-CMs, both patient lines demonstrated a dramatic reduction in caffeine and isoproterenol (ISO) stimulated Ca2+ transient amplitude, suggesting RyR2 loss-of-function. There was a >50% reduction in RYR2 transcript/RyR2 protein expression in both patient iPSC-CMs compared to WT. Delayed afterdepolarization was observed in the RYR2-DUP-iPSC-CMs but not in the WT-iPSC-CMs. Compared to WT-iPSC-CMs, there was a significantly elevated arrhythmic activity in the RYR2-DUP-iPSC-CMs in response to ISO. Nadolol, propranolol, and flecainide reduced erratic activity by 8.5 fold, 6.8 fold, and 2.4 fold, respectively from ISO challenge. Unlike the gain-of-function mechanism observed in RYR2-mediated CPVT, the homozygous multi-exon duplication precipitates a dramatic reduction in RYR2 transcription and RyR2 protein translation, a loss-of-function in calcium handling, and a calcium-induced calcium release apparatus that is insensitive to catecholamines and caffeine.
Authors
David J. Tester, CS John Kim, Samantha K. Hamrick, Dan Ye, Bailey J. O'Hare, Hannah M. Bombei, Kristi K. Fitzgerald, Carla M. Haglund-Turnquist, Dianne L. Atkins, Luis A. Ochoa Nunez, Ian H. Law, Joel D. Temple, Michael J. Ackerman
Abstract
Mutations in PKD1 (encoding for Polycystin-1, PC1) are found in 80-85% of patients with ADPKD. We tested the hypothesis that changes in actin dynamics result from PKD1 mutations through dysregulation of compartmentalised centrosomal RhoA signalling mediated by specific RhoGAP (ARHGAP) proteins resulting in the complex cellular cystic phenotype. Initial studies revealed that the actin cytoskeleton was highly disorganised in PKD1 patient-derived cells and was associated with an increase in total and centrosomal RhoA activation and ROCK signalling. Using cilia length as a phenotypic readout for centrosomal RhoA activity, we identified ARHGAP5, 29, 35 as essential regulators of ciliation in normal human renal tubular cells. Importantly, a specific decrease in centrosomal ARHGAP35 was observed in PKD1 null cells using a centrosome-targeted proximity ligation assay and by immunofluorescence labelling. Finally, we demonstrate that another ROCK inhibitor (hydroxyfasudil) reduced cyst expansion in both human PKD1 3D cyst assays and an inducible Pkd1 mouse model. In summary, we report a novel interaction between PC1 and ARHGAP35 in the regulation of centrosomal RhoA activation and ROCK signalling. Targeting the RhoA/ROCK pathway inhibited cyst formation in vitro and in vivo indicating its relevance to ADPKD pathogenesis and for developing new therapies to inhibit cyst initiation.
Authors
Andrew J. Streets, Philipp P. Prosseda, Albert C.M. Ong
Abstract
Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disorder caused by a polyglutamine expansion in the androgen receptor (AR). Using gene expression analysis and ChIP sequencing, we mapped transcriptional changes in genetically engineered patient stem cell–derived motor neurons. We found that transcriptional dysregulation in SBMA can occur through AR-mediated histone modification. We detected reduced histone acetylation, along with decreased expression of genes encoding compensatory metabolic proteins and reduced substrate availability for mitochondrial function. Furthermore, we found that pyruvate supplementation corrected this deficiency and improved mitochondrial function and SBMA motor neuron viability. We propose that epigenetic dysregulation of metabolic genes contributes to reduced mitochondrial ATP production. Our results show a molecular link between altered epigenetic regulation and mitochondrial metabolism that contributes to neurodegeneration.
Authors
Naemeh Pourshafie, Ester Masati, Eric Bunker, Alec R. Nickolls, Parisorn Thepmankorn, Kory Johnson, Xia Feng, Tyler Ekins, Christopher Grunseich, Kenneth H. Fischbeck
Abstract
Background: A treatment option for ADPKD has highlighted the need to identify rapidly progressive patients. Kidney size/age and genotype have predictive power for renal outcomes, but their relative and additive value, plus associated trajectories of disease progression, are not well defined. Methods: The value of genotypic and/or kidney imaging data (Mayo Imaging Class) to predict the time to functional (end stage kidney disease; ESKD, or decline in estimated glomerular filtration rate; eGFR) or structural (increase in height adjusted total kidney volume; htTKV) outcomes were evaluated in a Mayo Clinic PKD1/PKD2 population; and eGFR and htTKV trajectories from 20-65 years of age modeled and independently validated in similarly defined CRISP and HALT PKD patients. Results: Both genotypic and imaging groups strongly predicted ESKD and eGFR endpoints, with genotype improving the imaging predictions, and vice versa; a multivariate model had strong discriminatory power (C statistic = 0.845). However, imaging but not genotypic groups predicted htTKV growth, although more severe genotypic and imaging groups had larger kidneys at a young age. The trajectory of eGFR decline was linear from baseline in the most severe genotypic and imaging groups, but curvilinear in milder groups. Imaging class trajectories differentiated htTKV growth rates; severe classes had rapid early growth and large kidneys but growth later slowed. Conclusions: The value of imaging, genotypic, and combined data to identify rapidly progressive patients was demonstrated, and reference values for clinical trials provided. Our data indicates that differences in kidney growth rates before adulthood significantly define patients with severe disease. Funding: NIDDK grants: Mayo DK058816, DK090728; CRISP DK056943, DK056956, DK056957, DK056961; HALT PKD DK062410, DK062408, DK062402, DK082230, DK062411, DK062401.
Authors
Sravanthi Lavu, Lisa E. Vaughan, Sarah R. Senum, Timothy L. Kline, Arlene B. Chapman, Ronald D. Perrone, Michal Mrug, William E. Braun, Theodore I. Steinman, Frederic F. Rahbari-Oskoui, Godela M. Brosnahan, Kyongtae T. Bae, Douglas Landsittel, Fouad T. Chebib, Alan S. L. Yu, Vicente E. Torres, Peter C. Harris
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