Genetics
Abstract
Ion channel-controlled cell volume regulation is of fundamental significance to the physiological function of sperm. In addition to volume regulation, LRRC8A-dependent volume-regulated anion channel (VRAC) activity is involved in cell cycle progression, insulin signaling, and cisplatin resistance. Nevertheless, the contribution of LRRC8A and its dependent VRAC activity in the germ cell lineage remain unknown. By utilizing a spontaneous Lrrc8a mouse mutation (c.1325delTG, p.F443*) and genetically engineered mouse models, we demonstrate that LRRC8A-dependent VRAC activity is essential for male germ cell development and fertility. Lrrc8a-null male germ cells undergo progressive degeneration independent of the apoptotic pathway during postnatal testicular development. Lrrc8a-deficient mouse sperm exhibit multiple morphological abnormalities of the flagella (MMAF), a feature commonly observed in the sperm of infertile human patients. Importantly, we identified a human patient with a rare LRRC8A hypomorphic mutation (c.1634G>A, p.Arg545His) possibly linked to Sertoli cell–only syndrome (SCOS), a male sterility disorder characterized by the loss of germ cells. Thus, LRRC8A is a critical factor required for germ cell development and volume regulation in the mouse, and it might serve as a novel diagnostic and therapeutic target for SCOS patients.
Authors
Jianqiang Bao, Carlos J. Perez, Jeesun Kim, Huan Zhang, Caitlin J. Murphy, Tewfik Hamidi, Jean Jaubert, Craig D. Platt, Janet Chou, Meichun Deng, Meng-Hua Zhou, Yuying Huang, Héctor Gaitán-Peñas, Jean-Louis Guénet, Kevin Lin, Yue Lu, Taiping Chen, Mark T. Bedford, Sharon Y.R. Dent, John H. Richburg, Raúl Estévez, Hui-Lin Pan, Raif S. Geha, Qinghua Shi, Fernando Benavides
Abstract
BACKGROUND. There is currently no clinical distinction between different TP53 mutations, despite increasing evidence that not all mutations have equally deleterious effects on the activity of the encoded tumor suppressor protein p53. The objective of this study was to determine whether these biological differences have clinical significance. METHODS. This retrospective cohort analysis included 2,074 patients with sporadic TP53 mutations (403 unique mutations) and 1,049 germline TP53 mutation carriers (188 unique mutations). Survival was projected by stratifying patients according to their p53 mutant–specific residual transcriptional activity scores. RESULTS. Pan-cancer survival analyses revealed a strong association between increased mutant p53 residual activity and improved survival in males with glioma and gastric adenocarcinoma (P = 0.002 and P = 0.02) that was not present in the female cohorts (P = 0.16 and P = 0.50). Male glioma and gastric cancer patients with TP53 mutations resulting in >5% transcriptional activity had 3.1-fold (95% CI, 2.4–3.8; P = 0.002; multivariate analysis hazard ratio [HR]) and 4.6-fold (95% CI, 3.7–5.6; P = 0.001; multivariate analysis HR) lower risk of death as compared with patients harboring inactive (0% activity) p53 mutants. The correlation between mutant p53 residual activity with survival was recapitulated in the dataset of germline TP53 mutation carriers (HR = 3.0, 95% CI, 2.7–3.4, P < 0.001 [females]; HR = 2.2, 95% CI, 1.8–2.6, P < 0.001 [males]), where brain and gastric tumors were more common among males (P < 0.001 and P = 0.001, respectively). CONCLUSION. The retention of mutant p53 transcriptional activity prognosticates superior survival for men with glioma and gastric adenocarcinoma harboring sporadic TP53 mutations. Among germline TP53 mutation carriers, increased residual transcriptional activity is correlated with prolonged lifetime cancer survival and delayed tumor onset, and males are more prone to develop brain and gastric tumors. FUNDING. Canadian Institutes of Health Research (no. 148556).
Authors
Nicholas W. Fischer, Aaron Prodeus, Jean Gariépy
Abstract
Site-1 protease (S1P), encoded by MBTPS1, is a serine protease in the Golgi. S1P regulates lipogenesis, endoplasmic reticulum (ER) function, and lysosome biogenesis in mice and in cultured cells. However, how S1P differentially regulates these diverse functions in humans has been unclear. In addition, no human disease with S1P deficiency has been identified. Here, we report a pediatric patient with an amorphic and a severely hypomorphic mutation in MBTPS1. The unique combination of these mutations results in a frequency of functional MBTPS1 transcripts of approximately 1%, a finding that is associated with skeletal dysplasia and elevated blood lysosomal enzymes. We found that the residually expressed S1P is sufficient for lipid homeostasis but not for ER and lysosomal functions, especially in chondrocytes. The defective S1P function specifically impairs activation of the ER stress transducer BBF2H7, leading to ER retention of collagen in chondrocytes. S1P deficiency also causes abnormal secretion of lysosomal enzymes due to partial impairment of mannose-6-phosphate–dependent delivery to lysosomes. Collectively, these abnormalities lead to apoptosis of chondrocytes and lysosomal enzyme–mediated degradation of the bone matrix. Correction of an MBTPS1 variant or reduction of ER stress mitigated collagen-trafficking defects. These results define a new congenital human skeletal disorder and, more importantly, reveal that S1P is particularly required for skeletal development in humans. Our findings may also lead to new therapies for other genetic skeletal diseases, as ER dysfunction is common in these disorders.
Authors
Yuji Kondo, Jianxin Fu, Hua Wang, Christopher Hoover, J. Michael McDaniel, Richard Steet, Debabrata Patra, Jianhua Song, Laura Pollard, Sara Cathey, Tadayuki Yago, Graham Wiley, Susan Macwana, Joel Guthridge, Samuel McGee, Shibo Li, Courtney Griffin, Koichi Furukawa, Judith A. James, Changgeng Ruan, Rodger P. McEver, Klaas J. Wierenga, Patrick M. Gaffney, Lijun Xia
Abstract
Treatment of individuals with cystic fibrosis (CF) has been transformed by small molecule therapies that target select pathogenic variants in the CF transmembrane conductance regulator (CFTR). To expand treatment eligibility, we stably expressed 43 rare missense CFTR variants associated with moderate CF from a single site in the genome of human CF bronchial epithelial (CFBE41o–) cells. The magnitude of drug response was highly correlated with residual CFTR function for the potentiator ivacaftor, the corrector lumacaftor, and ivacaftor-lumacaftor combination therapy. Response of a second set of 16 variants expressed stably in Fischer rat thyroid (FRT) cells showed nearly identical correlations. Subsets of variants were identified that demonstrated statistically significantly higher responses to specific treatments. Furthermore, nearly all variants studied in CFBE cells (40 of 43) and FRT cells (13 of 16) demonstrated greater response to ivacaftor-lumacaftor combination therapy than either modulator alone. Together, these variants represent 87% of individuals in the CFTR2 database with at least 1 missense variant. Thus, our results indicate that most individuals with CF carrying missense variants are (a) likely to respond modestly to currently available modulator therapy, while a small fraction will have pronounced responses, and (b) likely to derive the greatest benefit from combination therapy.
Authors
Sangwoo T. Han, Andras Rab, Matthew J. Pellicore, Emily F. Davis, Allison F. McCague, Taylor A. Evans, Anya T. Joynt, Zhongzhou Lu, Zhiwei Cai, Karen S. Raraigh, Jeong S. Hong, David N. Sheppard, Eric J. Sorscher, Garry R. Cutting
Abstract
Mice are extremely important as the premier model organism in human biomedical and mammalian genetic research. The genomes of several tens of mouse inbred strains have been sequenced. They have been compared to the genome of C57BL/6J, considered by convention as the reference genome. Based on a comparison of this reference genome with 36 other sequenced mouse strains, we generated an overview of all protein-coding genes that are deviant in this reference genome, compared with consensus protein-coding mouse gene sequences. We provide PROVEAN scores, reflecting the likelihood that these C57BL/6J proteins have lost function. We thus identified numerous abnormal proteins, and biological pathways, specifically present in C57BL/6J, suggesting the important caveats of this reference mouse strain, and linking candidate genes to some of the best-known phenotypes of this strain.
Authors
Steven Timmermans, Claude Libert
Abstract
Numerous studies of relatively few patients have linked T cell receptor (TCR) genes to psoriasis but have yielded dramatically conflicting results. To resolve these discrepancies, we have chosen to mine RNA-Seq datasets for patterns of TCR gene segment usage in psoriasis. A meta-analysis of 3 existing and 1 unpublished datasets revealed a statistically significant link between the relative expression of TRAJ23 and psoriasis and the psoriasis-associated cytokine IL-17A. TRGV5, a TCR-γ segment, was also associated with psoriasis but correlated instead with IL-36A, other IL-36 family members, and IL-17C (not IL-17A). In contrast, TRAJ39 was strongly associated with healthy skin. T cell diversity measurements and analysis of CDR3 sequences were also conducted, revealing no psoriasis-associated public CDR3 sequences. Finally, in comparison with the expression of TCR-αβ genes, the expression of TCR-γδ genes was relatively low but mildly elevated in psoriatic skin. These results have implications for the development of targeted therapies for psoriasis and other autoimmune diseases. Also, the techniques employed in this study have applications in other fields, such as cancer immunology and infectious disease.
Authors
Alexander A. Merleev, Alina I. Marusina, Chelsea Ma, James T. Elder, Lam C. Tsoi, Siba P. Raychauduri, Stephan Weidinger, Elizabeth A. Wang, Iannis E. Adamopoulos, Guillaume Luxardi, Johann E. Gudjonsson, Michiko Shimoda, Emanual Maverakis
Abstract
Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2’-O-methyl phosphorothioate oligonucleotides and vivo–phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo–phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.
Authors
Eugene J. Wyatt, Alexis R. Demonbreun, Ellis Y. Kim, Megan J. Puckelwartz, Andy H. Vo, Lisa M. Dellefave-Castillo, Quan Q. Gao, Mariz Vainzof, Rita C. M. Pavanello, Mayana Zatz, Elizabeth M. McNally
Abstract
Mutations in the gene encoding the protein kinase A (PKA) catalytic subunit α have been found to be responsible for cortisol-producing adenomas (CPAs). In this study, we identified by whole-exome sequencing the somatic mutation p.S54L in the PRKACB gene, encoding the catalytic subunit β (Cβ) of PKA, in a CPA from a patient with severe Cushing syndrome. Bioluminescence resonance energy transfer and surface plasmon resonance assays revealed that the mutation hampers formation of type I holoenzymes and that these holoenzymes were highly sensitive to cAMP. PKA activity, measured both in cell lysates and with recombinant proteins, based on phosphorylation of a synthetic substrate, was higher under basal conditions for the mutant enzyme compared with the WT, while maximal activity was lower. These data suggest that at baseline the PRKACB p.S54L mutant drove the adenoma cells to higher cAMP signaling activity, probably contributing to their autonomous growth. Although the role of PRKACB in tumorigenesis has been suggested, we demonstrated for the first time to our knowledge that a PRKACB mutation can lead to an adrenal tumor. Moreover, this observation describes another mechanism of PKA pathway activation in CPAs and highlights the particular role of residue Ser54 for the function of PKA.
Authors
Stéphanie Espiard, Matthias J. Knape, Kerstin Bathon, Guillaume Assié, Marthe Rizk-Rabin, Simon Faillot, Windy Luscap-Rondof, Daniel Abid, Laurence Guignat, Davide Calebiro, Friedrich W. Herberg, Constantine A. Stratakis, Jérôme Bertherat
Abstract
Heterotopic ossification (HO) is a significant clinical problem with incompletely resolved mechanisms. Here, the secreted metalloproteinases ADAMTS7 and ADAMTS12 are shown to comprise a unique proteoglycan class that protects against a tendency toward HO in mouse hindlimb tendons, menisci, and ligaments. Adamts7 and Adamts12 mRNAs were sparsely expressed in murine forelimbs but strongly coexpressed in hindlimb tendons, skeletal muscle, ligaments, and meniscal fibrocartilage. Adamts7–/– Adamts12–/– mice, but not corresponding single-gene mutants, which demonstrated compensatory upregulation of the intact homolog mRNA, developed progressive HO in these tissues after 4 months of age. Adamts7–/– Adamts12–/– tendons had abnormal collagen fibrils, accompanied by reduced levels of the small leucine-rich proteoglycans (SLRPs) biglycan, fibromodulin, and decorin, which regulate collagen fibrillogenesis. Bgn–/0 Fmod–/– mice are known to have a strikingly similar hindlimb HO to that of Adamts7–/– Adamts12–/– mice, implicating fibromodulin and biglycan reduction as a likely mechanism underlying HO in Adamts7–/– Adamts12–/– mice. Interestingly, degenerated human biceps tendons had reduced ADAMTS7 mRNA compared with healthy biceps tendons, which expressed both ADAMTS7 and ADAMTS12. These results suggest that ADAMTS7 and ADAMTS12 drive an innate pathway protective against hindlimb HO in mice and may be essential for human tendon health.
Authors
Timothy J. Mead, Daniel R. McCulloch, Jason C. Ho, Yaoyao Du, Sheila M. Adams, David E. Birk, Suneel S. Apte
Abstract
BACKGROUND. HELLP (hemolysis, elevated liver enzymes, and low platelets) syndrome is a severe variant of hypertensive disorders of pregnancy affecting approximately 1% of all pregnancies, and has significant maternal and fetal morbidity. Previously, we showed that upregulation of the alternative pathway of complement (APC) plays a role in HELLP syndrome. We hypothesize that HELLP syndrome follows a 2-hit disease model similar to atypical hemolytic uremic syndrome (aHUS), requiring both genetic susceptibility and an environmental risk factor. Our objective was to perform a comparative analysis of the frequency of APC activation and germline mutations in affected women and to create a predictive model for identifying HELLP syndrome. METHODS. Pregnant women with HELLP syndrome, and healthy controls after 23 weeks of gestation were recruited, along with aHUS and thrombotic thrombocytopenic purpura participants. We performed a functional assay, the mHam, and targeted genetic sequencing in all groups. RESULTS. Significantly more participants with rare germline mutations in APC genes were present in the HELLP cohort compared with controls (46% versus 8%, P = 0.01). In addition, significantly more HELLP participants were positive for the mHam when compared with controls (62% versus 16%, P = 0.009). Testing positive for both a germline mutation and the mHam was highly predictive for the diagnosis of HELLP syndrome. CONCLUSION. HELLP syndrome is characterized by both activation of the APC and frequent germline mutations in APC genes. Similar to aHUS, treatment via complement inhibition to mitigate maternal and fetal morbidity and mortality may be possible. FUNDING. National Heart Lung and Blood Institute grants T32HL007525 and R01HL133113.
Authors
Arthur J. Vaught, Evan M. Braunstein, Jagar Jasem, Xuan Yuan, Igor Makhlin, Solange Eloundou, Andrea C. Baines, Samuel A. Merrill, Shruti Chaturvedi, Karin Blakemore, C. John Sperati, Robert A. Brodsky
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