Genetics
Abstract
Hereditary Macular Dystrophies (HMDs) are a genetically diverse group of disorders that cause central vision loss due to photoreceptor and retinal pigment epithelium (RPE) damage. We investigated a family with a presumed novel autosomal dominant HMD characterized by faint, hypopigmented RPE changes involving the central retina. Genome and RNA sequencing identified the disease-causing variant to be a 560 kilobase tandem duplication on chromosome 17 [NC_000017.10 (hg19): g.4012590_4573014dup], which led to the formation of a novel ZZEF1-ALOX15 fusion gene, that upregulates ALOX15. ALOX15 encodes a lipoxygenase involved in polyunsaturated fatty acid metabolism. Functional studies showed retinal disorganization, and photoreceptor and RPE damage following electroporation of the chimera transcript in mouse retina. Photoreceptor damage also occurred following electroporation with a native ALOX15 transcript but not with a near-null ALOX15 transcript. Affected patients’ lymphoblasts demonstrated lower levels of ALOX15 substrates and an accumulation of neutral lipids. We implicated the fusion gene as the cause of this family’s HMD, due to mis-localization and overexpression of ALOX15, driven by the ZZEF1 promoter. To our knowledge, this is the first reported instance of a fusion gene leading to HMD or inherited retinal dystrophy, highlighting the need to prioritize duplication analysis in unsolved retinal dystrophies.
Authors
Rabiat Adele, Rowaida Hussein, Erika Tavares, Kashif Ahmed, Matteo Di Scipio, Jason Charish, Minggao Liang, Simon Monis, Anupreet Tumber, Xiaoyan Chen, Tara A. Paton, Nicole M. Roslin, Christabel Eileen, Evgueni Ivakine, Nishanth E. Sunny, Michael D. Wilson, Eric Campos, Raju V.S. Rajala, Jason T. Maynes, Philippe P. Monnier, Andrew D. Paterson, Elise Héon, Ajoy Vincent
Abstract
Graves' disease (GD) is an autoimmune condition that can progress to Graves' Ophthalmopathy (GO), leading to irreversible damage to orbital tissues and potential blindness. The pathogenic mechanism is not fully understood. In this study, we conducted single-cell multi-omics analyses on healthy individuals, GD patients without GO, newly diagnosed GO patients, and treated GO patients. Our findings revealed gradual systemic inflammation during GO progression, marked by overactivation of cytotoxic effector T cell subsets, and expansion of specific T cell receptor clones. Importantly, we observed a decline in the immunosuppressive function of activated regulatory T cells (aTreg) accompanied by a cytotoxic phenotypic transition. In vitro experiments revealed that dysfunction and transition of GO-autoreactive Treg were regulated by the yinyang1 (YY1) upon secondary stimulation of thyroid stimulating hormone receptor (TSHR) under inflammatory conditions. Furthermore, adoptive transfer experiments of GO mouse model confirmed infiltration of these cytotoxic Treg into the orbital lesion tissues. Notably, these cells were found to upregulate inflammation and promote pathogenic fibrosis of orbital fibroblasts (OFs). Our results revealed the dynamic changes in immune landscape during GO progression and provided novel insights into the instability and phenotypic transition of Treg, offering potential targets for therapeutic intervention and prevention of autoimmune diseases.
Authors
Zhong Liu, Shurui Ke, Zhuoxing Shi, Ming Zhou, Li Sun, Qihang Sun, Bing Xiao, Dongliang Wang, Yanjing Huang, Jinshan Lin, Huishi Wang, Qikai Zhang, Caineng Pan, Xuanwei Liang, Rongxin Chen, Zhen Mao, Xianchai Lin
Abstract
Leucine-zipper-like post translational regulator 1 (LZTR1) is a member of the BTB-Kelch superfamily, which regulates the RAS proteostasis. Autosomal dominant (AD) mutations in LZTR1 have been identified in patients with Noonan syndrome (NS), a congenital anomaly syndrome. However, it remains unclear whether LZTR1 AD mutations regulate the proteostasis of the RAS subfamily molecules or cause NS-like phenotypes in vivo. To elucidate the pathogenesis of LZTR1 mutations, we generated two novel LZTR1 mutation knock-in mice (Lztr1G245R/+ and Lztr1R409C/+), which correspond to the human p.G248R and p.R412C mutations, respectively. LZTR1-mutant male mice exhibit low birth weight, distinctive facial features, and cardiac hypertrophy. Cardiomyocyte size and the expression of RAS subfamily members, including MRAS and RIT1, were significantly increased in the left ventricles (LVs) of mutant male mice. LZTR1 AD mutants did not interact with RIT1 and functioned as dominant-negative forms of wild-type LZTR1. Multi-omics analysis revealed that the MAPK signaling pathway was activated in the LVs of mutant mice. Treatment with the MEK inhibitor trametinib ameliorated cardiac hypertrophy in mutant male mice. These results suggest that MEK/ERK pathway is a therapeutic target for NS-like phenotype resulting from dysfunction of RAS proteostasis by LZTR1 AD mutations.
Authors
Taiki Abe, Kaho Morisaki, Tetsuya Niihori, Miho Terao, Shuji Takada, Yoko Aoki
Abstract
Multiple sclerosis (MS) is a complex disease with significant heterogeneity in disease course and progression. Genetic studies have identified numerous loci associated with MS risk, but the genetic basis of disease progression remains elusive. To address this, we leveraged the Collaborative Cross (CC), a genetically diverse mouse strain panel, and experimental autoimmune encephalomyelitis (EAE). The thirty-two CC strains studied captured a wide spectrum of EAE severity, trajectory, and presentation, including severe-progressive, monophasic, relapsing remitting, and axial rotary (AR)-EAE, accompanied by distinct immunopathology. Sex differences in EAE severity were observed in six strains. Quantitative trait locus analysis revealed distinct genetic linkage patterns for different EAE phenotypes, including EAE severity and incidence of AR-EAE. Machine learning-based approaches prioritized candidate genes for loci underlying EAE severity (Abcc4 and Gpc6) and AR-EAE (Yap1 and Dync2h1). This work expands the EAE phenotypic repertoire and identifies novel loci controlling unique EAE phenotypes, supporting the hypothesis that heterogeneity in MS disease course is driven by genetic variation.
Authors
Emily A. Nelson, Anna L. Tyler, Taylor Lakusta-Wong, Karolyn G. Lahue, Katherine C. Hankes, Cory Teuscher, Rachel M. Lynch, Martin T. Ferris, J. Matthew Mahoney, Dimitry N. Krementsov
Abstract
Gaucher disease, the most prevalent lysosomal storage disease, is caused by homozygous mutations at the GBA gene, responsible for encoding the enzyme glucocerebrosidase. Neuronopathic Gaucher disease is associated with microgliosis, astrogliosis, and neurodegeneration. However, the role that microglia, astrocytes, and neurons play in the disease remains to be determined. In the current study, we developed novel, inducible, cell-type specific GBA KO mice to understand the individual impacts of GBA deficiencies on microglia and neurons. GBA was conditionally knocked out either exclusively in microglia or neurons, or throughout the body. These novel mouse models were developed using a tamoxifen-inducible Cre system, with tamoxifen administration commencing at weaning. Microglia-specific GBA KO mice showed no signs of disease. However, the neuron-specific GBA KO resulted in a shortened lifespan, severe weight loss, and ataxia. These mice also had significant neurodegeneration, microgliosis, and astrogliosis accompanied by the accumulation of glucosylceramide and glucosylsphingosine, recapitulating Gaucher disease-like symptoms. These surprising findings reveal that, unlike the neuron-specific GBA deficiency, microglia-specific GBA deficiency alone does not induce disease. The novel neuronal Gaucher disease mouse model, with a median survival of 16 weeks, may be useful for future studies of pathogenesis and the evaluation of therapies.
Authors
Hannah B. D. Duffy, Colleen Byrnes, Hongling Zhu, Galina Tuymetova, Y. Terry Lee, Frances M. Platt, Richard L. Proia
Abstract
Spinal muscular atrophy (SMA) is a recessive, developmental disorder caused by the genetic loss or mutation of the gene SMN1 (Survival of Motor Neuron 1). SMA is characterized by neuromuscular symptoms and muscle weakness. Several years ago, SMA treatment underwent a radical transformation, with the approval of three different SMN-dependent disease modifying therapies. This includes two SMN2 splicing therapies - Risdiplam and Nusinersen. One main challenge for Type II SMA patients treated with these drugs is ongoing muscle fatigue, limited mobility, and other skeletal problems. To date, few molecular studies have been conducted on SMA-patient derived tissues after treatment, limiting our understanding of what targets remain after the principal spinal cord targeted therapies are applied. Therefore, we collected paravertebral muscle from eight Type II patients undergoing spinal surgery for scoliosis and seven controls. We used RNA-sequencing to characterize their transcriptional profiles and correlate these with muscle histology. Despite the limited cohort size and heterogeneity, we observed a consistent loss of oxidative phosphorylation machinery of the mitochondria, a decrease in mitochondrial DNA copy number, and a correlation between signals of cellular stress, denervation and increased fibrosis. This work provides new putative targets for combination therapies for Type II SMA.
Authors
Fiorella Grandi, Stéphanie Astord, Sonia Pezet, Elèna Gidaja, Sabrina Mazzucchi, Maud Chapart, Stéphane Vasseur, Kamel Mamchaoui, Piera Smeriglio
Abstract
Despite advances in sequencing technologies, a molecular diagnosis remains elusive in many Mendelian disease patients. Current short-read clinical sequencing approaches cannot provide chromosomal phase information or epigenetic information without further sample processing, which is not routinely done and can result in an incomplete molecular diagnosis in patients. The ability to provide phased genetic and epigenetic information from a single sequencing run would improve the diagnostic rate of Mendelian conditions. Here we describe Targeted Long-read Sequencing of Mendelian Disease genes (TaLon-SeqMD) using a real-time adaptive sequencing approach. Optimization of bioinformatic targeting enabled selective enrichment of multiple disease-causing regions of the human genome. Haplotype-resolved variant calling and simultaneous resolution of epigenetic base modification could be achieved in a single sequencing run. The TaLon-SeqMD approach was validated in a cohort of 18 subjects with previous genetic testing targeting 373 inherited retinal disease (IRD) genes, yielding the complete molecular diagnosis in each case. This approach was then applied in two IRD cases with inconclusive testing, which uncovered non-coding and structural variants that were difficult to characterize by standard short-read sequencing. Overall, these results demonstrate TaLon-SeqMD as an approach to provide rapid phased-variant calling to provide the molecular basis of Mendelian diseases.
Authors
Kenji Nakamichi, Jennifer Huey, Riccardo Sangermano, Emily M. Place, Kinga M. Bujakowska, Molly Marra, Lesley A. Everett, Paul Yang, Jennifer R. Chao, Russell N. Van Gelder, Debarshi Mustafi
Abstract
Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto acid dehydrogenase complexes. DLD disease symptoms are multi-systemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, understanding the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.
Authors
Manuela Lavorato, Donna Iadarola, Cristina Remes, Prabhjot Kaur, Chynna Broxton, Neal D. Mathew, Rui Xiao, Christoph Seiler, Eiko Nakamaru-Ogiso, Vernon E. Anderson, Marni J. Falk
Abstract
This study aimed at defining the role of the B-cell adaptor protein BANK1 in the appearance of age-associated B cells (ABCs) in two SLE mouse models (TLR7.tg6 and Imiquimod-induced mice), crossed with Bank1-/- mice. The absence of Bank1 led to a significant reduction in ABC levels, also affecting other B cell populations. To gain deeper insights into their differentiation pathway and the impact of Bank1 on B cell populations, a single-cell transcriptome assay was performed. In the TLR7.tg6 model, we identified 10 clusters within B cells, including an ABC-specific cluster which was decreased in Bank1-deficient mice. In its absence, ABCs exhibited an anti-inflammatory gene expression profile, while being pro-inflammatory in Bank1-sufficient lupus mice. Trajectory analyses revealed that ABCs originated from marginal zone and memory-like B cells, ultimately acquiring transcriptional characteristics associated with atypical memory cells and long-lived plasma cells. Also, Bank1 deficiency normalized the presence of naïve B cells, which were nearly absent in lupus mice. Interestingly, Bank1 deficiency significantly reduced a distinct cluster containing IFN-responsive genes. These findings underscore the critical role of Bank1 in ABC development, impacting early B cell stages towards ABC differentiation, and the presence of IFN-stimulated gene-containing B cells, both populations determinant for autoimmunity.
Authors
Gonzalo Gómez Hernández, Daniel Toro-Domínguez, Georgina Galicia, María Morell, Marta E. Alarcón-Riquelme
Abstract
BACKGROUND. Two coding alleles within the APOL1 gene, G1 and G2, found almost exclusively in individuals genetically similar to West African populations, contribute substantially to the pathogenesis of chronic kidney disease (CKD). The APOL gene cluster on chromosome 22 contains a total of six APOL genes that have arisen as a result of gene duplication. METHODS. Using a genome-first approach in the Penn Medicine Biobank, we identified 62 protein-altering variants in the six APOL genes with a minor allele frequency > 0.1% in a population of participants genetically similar to African reference populations and performed population-specific phenome-wide association studies. RESULTS. We identified rs1108978, a stop-gain variant in APOL3 (p.Q58*), to be significantly associated with increased CKD risk, even after conditioning on APOL1 G1/G2 carrier status. These findings were replicated in the Veterans Affairs Million Veteran Program and the All of Us Research Program. APOL3 p.Q58* was also significantly associated with a number of quantitative traits linked to CKD including decreased kidney volume. This truncating variant contributed the most risk for CKD in patients monoallelic for APOL1 G1/G2, suggesting an epistatic interaction and a potential protective effect of wild-type APOL3 against APOL1-induced kidney disease. CONCLUSION. This study demonstrates the utility of targeting population-specific variants in a genome-first approach, even in the context of well-studied gene-disease relationships. FUNDING. National Heart, Lung, and Blood Institute (F30HL172382, R01HL169378, R01HL169458), Doris Duke Foundation (grant 2023-0224), National Institute of Biomedical Imaging and Bioengineering (P41EB029460), National Center for Advancing Translational Sciences (UL1-TR-001878).
Authors
David Y. Zhang, Michael G. Levin, Jeffrey T. Duda, Latrice G. Landry, Walter R. Witschey, Scott M. Damrauer, Marylyn D. Ritchie, Daniel J. Rader
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