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Genetics

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Multimodal single-cell analysis of non-random heteroplasmy distribution in human retinal mitochondrial disease
Nathaniel K. Mullin, … , Budd A. Tucker, Robert F. Mullins
Nathaniel K. Mullin, … , Budd A. Tucker, Robert F. Mullins
Published June 8, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.165937.
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Multimodal single-cell analysis of non-random heteroplasmy distribution in human retinal mitochondrial disease

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Abstract

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multi-system disease. The wide range of manifestations observed in mitochondrial disease patients results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify non-random distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA sequencing, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profile the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and healthy control donors. Utilizing the retina as a model for complex multi-lineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the wildtype allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the non-random nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

Authors

Nathaniel K. Mullin, Andrew P. Voigt, Miles J. Flamme-Wiese, Xiuying Liu, Megan J. Riker, Katayoun Varzavand, Edwin M. Stone, Budd A. Tucker, Robert F. Mullins

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High-fat diet plus HNF1A variant promotes polyps by activating β-catenin in early onset colorectal cancer
Heyu Song, … , Elena M. Stoffel, Juanita L. Merchant
Heyu Song, … , Elena M. Stoffel, Juanita L. Merchant
Published May 23, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.167163.
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High-fat diet plus HNF1A variant promotes polyps by activating β-catenin in early onset colorectal cancer

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Abstract

The incidence of early onset colorectal cancer (EO-CRC) is rising and is poorly understood. Lifestyle factors and altered genetic background possibly contribute. Here we performed targeted exon sequencing of archived leukocyte DNA from 158 EO-CRC participants, which identified a missense mutation at p.A98V within the proximal DNA binding domain of Hepatic Nuclear Factor 1 alpha (HNF1AA98V, Rs1800574). The HNF1AA98V exhibited reduced DNA binding. To test function, the HNF1A variant was introduced into the mouse genome by CRISPR/Cas9 and the mice were placed on either a high fat (HFD) or high sugar diet (HSD). Only 1% of the HNF1A mutant mice developed polyps on normal chow; however,19% and 3% developed polyps on the HFD and HSD, respectively. RNA-Seq revealed an increase in metabolic, immune, lipid biogenesis genes and Wnt/β-catenin signaling components in the HNF1A mutant relative to the wildtype mice. Mouse polyps and colon cancers from subjects carrying the HNF1AA98V variant exhibited reduced CDX2 and elevated β-catenin proteins. We further demonstrated decreased occupancy of HNF1AA98V at the Cdx2 locus and reduced Cdx2 promoter activity compared to wildtype HNF1A. Collectively, our study shows that the HNF1AA98V variant plus HFD promotes the formation of colonic polyps by activating β-catenin via decreasing Cdx2 expression.

Authors

Heyu Song, Ricky A. Sontz, Matthew J. Vance, Julia M. Morris, Sulaiman Sheriff, Songli Zhu, Suzann Duan, Jiping Zeng, Erika Koeppe, Ritu Pandey, Curtis A. Thorne, Elena M. Stoffel, Juanita L. Merchant

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Lymphatic disorders caused by mosaic, activating KRAS variants respond to MEK inhibition
Sarah E. Sheppard, … , Yoav Dori, Hakon Hakonarson
Sarah E. Sheppard, … , Yoav Dori, Hakon Hakonarson
Published May 8, 2023
Citation Information: JCI Insight. 2023;8(9):e155888. https://doi.org/10.1172/jci.insight.155888.
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Lymphatic disorders caused by mosaic, activating KRAS variants respond to MEK inhibition

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Abstract

Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2‑dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.

Authors

Sarah E. Sheppard, Michael E. March, Christoph Seiler, Leticia S. Matsuoka, Sophia E. Kim, Charlly Kao, Adam I. Rubin, Mark R. Battig, Nahla Khalek, Erica Schindewolf, Nora O’Connor, Erin Pinto, Jessica R.C. Priestley, Victoria R. Sanders, Rojeen Niazi, Arupa Ganguly, Cuiping Hou, Diana Slater, Ilona J. Frieden, Thy Huynh, Joseph T. Shieh, Ian D. Krantz, Jessenia C. Guerrero, Lea F. Surrey, David M. Biko, Pablo Laje, Leslie Castelo-Soccio, Taizo A. Nakano, Kristen Snyder, Christopher L. Smith, Dong Li, Yoav Dori, Hakon Hakonarson

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The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes
Amjad Horani, … , Susan K. Dutcher, Steven L. Brody
Amjad Horani, … , Susan K. Dutcher, Steven L. Brody
Published April 27, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.168836.
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The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

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Abstract

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies.

Authors

Amjad Horani, Deepesh Gupta, Jian Xu, Huihui Xu, Lis del C. Puga Molina, Celia M. Santi, Sruthi Ramagiri, Steven K. Brennan, Jiehong Pan, Jeffrey R. Koenitzer, Tao Huang, Rachael M. Hyland, Sean P. Gunsten, Shin-Cheng Tzeng, Jennifer M. Strahle, Pleasantine Mill, Moe R. Mahjoub, Susan K. Dutcher, Steven L. Brody

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Multi-ancestry sex-stratified genomic associations with HIV viral load and controller status from the ICGH
Candelaria Vergara, … , Eileen P. Scully, Paul J. McLaren
Candelaria Vergara, … , Eileen P. Scully, Paul J. McLaren
Published April 25, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.170068.
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Multi-ancestry sex-stratified genomic associations with HIV viral load and controller status from the ICGH

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Abstract

Biological sex and host genetics influence HIV pathogenesis. Females have a higher likelihood of spontaneous viral control and lower setpoint viral load (spVL). No prior studies have assessed sex-specific genetics of HIV. To address this, we performed a sex stratified genome-wide association study using data from the International Collaboration for the Genomics of HIV. Although it is the largest collection of genomic data in HIV, this multi-ethnic sample of 9,705 people is 81.3% male. We sought to identify sex-specific genetic variants and genes associated with HIV spVL and control. We confirmed associations in the HLA and CCR5 regions in males, and HLA in females. Gene-based analyses detected associations between HIV spVL and PET100 (Pvalue=8.36x10-07), PCP2 (Pvalue=8.81x10-07), XAB2 (Pvalue=1.32x10-6) and STXBP2 (Pvalue=1.65x10-4) only in males. We detected variants with a significant sex-differential effect on spVL in SDC3 and PUM1 (rs10914268,Pvalue=1.93x10-08) and PSORS1C2 (rs1265159, Pvalue=3.26x10-08) and on HIV control in SUB1 (rs687659, Pvalue=1.02×10-08), AL158151.3, PTPA and IER5L (rs4387067, Pvalue=2.07×10-09). Those variants have epigenetic and genetic interactions with relevant genes with both cis and trans effects. In summary, we identified sex-shared associations at the single variant level, sex-specific associations at the gene-based level, and genetic variants with significant differential effects between the sexes.

Authors

Candelaria Vergara, Jeffrey F. Tuff, International Collaboration for the Genomics of HIV, Jacques Fellay, Priya Duggal, Eileen P. Scully, Paul J. McLaren

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Gene augmentation of LCA5-Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme
Siebren Faber, … , Virginie Hamel, Ronald Roepman
Siebren Faber, … , Virginie Hamel, Ronald Roepman
Published April 18, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.169162.
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Gene augmentation of LCA5-Leber congenital amaurosis ameliorates bulge region defects of the photoreceptor ciliary axoneme

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Abstract

Leber congenital amaurosis (LCA) is a group of inherited retinal diseases (IRDs) characterized by the early onset and rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy (U-ExM), we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localize at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient for lebercilin exhibit early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, AAV-based LCA5 gene augmentation partially restores the bulge region, preserves OS axoneme structure and membrane disc formation, and results in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.

Authors

Siebren Faber, Olivier Mercey, Katrin Junger, Alejandro Garanto, Marius Ueffing, Rob W.J. Collin, Karsten Boldt, Paul Guichard, Virginie Hamel, Ronald Roepman

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An SNX31 variant underlies dominant familial exudative vitreoretinopathy-like pathogenesis
Ningda Xu, … , Jing Li, Lvzhen Huang
Ningda Xu, … , Jing Li, Lvzhen Huang
Published April 13, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.167032.
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An SNX31 variant underlies dominant familial exudative vitreoretinopathy-like pathogenesis

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Abstract

Familial exudative vitreoretinopathy (FEVR) is a complex hereditary eye disorder characterized by incomplete development of the retinal vasculature, thereby affecting retinal angiogenesis. But the genetic factors contributing to its development or pathogenesis remain elusive. In a Chinese FEVR family with 19 members, by utilizing whole exome sequencing, we identified a candidate disease-causing DNA variant in sorting nexin 31 (SNX31) (c.963delG; p. Trp321Cys), which results in a frameshift mutation. Herein we studied the biochemical mechanism of this mutation and uncovered that it is deficient in β1-integrin binding and integrin stability. The SNX31 c.963delG point mutation mouse model (SNX31m/m) was constructed using CRISPR/Cas9 technology. At 2-4 months of age, SNX31m/m mice showed fundus phenotypes similar to FEVR-like changes, including vascular leakage and retinal atrophy. Moreover, we found that VEGF and apoptotic pathways were involved in these ocular phenotypes. At present, the FEVR-like mouse model is mainly constructed by intravitreal injection, and we are the first to construct it by gene knockout. Hence our study extended FEVR mutation spectrum to include SNX31. Meanwhile, these findings expanded our understanding of the molecular pathogenesis of FEVR and may facilitate the development of methods for the diagnosis and prevention of FEVR patients.

Authors

Ningda Xu, Yi Cai, Jiarui Li, Tianchang Tao, Caifei Liu, Yan Shen, Xiaoxin Li, Leiliang Zhang, Mingwei Zhao, Xuan Shi, Jing Li, Lvzhen Huang

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Multi-omics of Bohring-Opitz syndrome truncating ASXL1 mutations identify canonical and non-canonical Wnt signaling dysregulation
Isabella Lin, … , Rosanna Weksberg, Valerie A. Arboleda
Isabella Lin, … , Rosanna Weksberg, Valerie A. Arboleda
Published April 13, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.167744.
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Multi-omics of Bohring-Opitz syndrome truncating ASXL1 mutations identify canonical and non-canonical Wnt signaling dysregulation

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Abstract

ASXL1 (Additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS, OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, characteristic facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies including heart defects and severe skeletal defects giving rise to a typical ‘BOS posture’. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia (AML). We use primary cells from BOS individuals (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multi-omics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data shows that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, a planar cell polarity pathway protein that acts through non-canonical Wnt signaling to direct tissue patterning and cell migration. This multi-omics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias.

Authors

Isabella Lin, Angela Wei, Zain Awamleh, Meghna Singh, Aileen Ning, Analeyla Herrera, Bianca E. Russell, Rosanna Weksberg, Valerie A. Arboleda

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Dissecting aortic aneurysm in Marfan syndrome is associated with losartan-sensitive transcriptomic modulation of aortic cells
Yifei Sun, … , Martin J. Walsh, Francesco Ramirez
Yifei Sun, … , Martin J. Walsh, Francesco Ramirez
Published April 6, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.168793.
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Dissecting aortic aneurysm in Marfan syndrome is associated with losartan-sensitive transcriptomic modulation of aortic cells

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Abstract

To improve our limited understanding of the pathogenesis of thoracic aortic aneurysm (TAA) leading to acute aortic dissection, single-cell RNA sequencing (scRNA-seq) was employed to profile disease-relevant transcriptomic changes of aortic cell populations in a well-characterized mouse model of the most commonly diagnosed form of Marfan syndrome (MFS). As result, two discrete sub-populations of aortic cells (SMC3 and EC4) were identified only in the aorta of Fbn1mgR/mgR mice. SMC3 highly express genes related to extracellular matrix formation and nitric oxide signaling, whereas EC4 transcriptional profile is enriched in SMC, fibroblast, and immune cell-related genes. Trajectory analysis predicted close phenotypic modulation between SMC3 and EC4, which were therefore analyzed together as a discrete MFS-modulated (MFSmod) sub-population. In situ hybridizations of diagnostic transcripts located MFSmod cells to the intima of Fbn1mgR/mgR aortas. Reference-based dataset integration revealed transcriptomic similarity between MFSmod and an SMC-derived cell cluster modulated in human TAA. Consistent with angiotensin II type I receptor (At1r) contribution to TAA development, MFSmod cells were absent in the aorta of Fbn1mgR/mgR mice treated with the At1r antagonist losartan. Altogether, our findings indicate that a discrete dynamic alteration of aortic cell identity is associated with dissecting TAA in MFS mice and increased risk of aortic dissection in MFS patients.

Authors

Yifei Sun, Keiichi Asano, Lauriane Sedes, Anna Cantalupo, Jens Hansen, Ravi Iyengar, Martin J. Walsh, Francesco Ramirez

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Loss of function variant in SPIN4 causes an X-linked overgrowth syndrome
Julian C. Lui, … , Youn Hee Jee, Jeffrey Baron
Julian C. Lui, … , Youn Hee Jee, Jeffrey Baron
Published March 16, 2023
Citation Information: JCI Insight. 2023. https://doi.org/10.1172/jci.insight.167074.
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Loss of function variant in SPIN4 causes an X-linked overgrowth syndrome

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Abstract

Overgrowth syndromes can be caused by pathogenic genetic variants in epigenetic writers, such as DNA and histone methyltransferases. However, no overgrowth disorder has previously been ascribed to variants in a gene that acts primarily as an epigenetic reader. Here, we studied a male individual with generalized overgrowth of prenatal onset. Exome sequencing identified a hemizygous frameshift variant in Spindlin 4 (SPIN4), with X-linked inheritance. We found evidence that SPIN4 binds specific histone modifications, promotes canonical WNT signaling, and inhibits cell proliferation in vitro and that the identified frameshift variant had lost all of these functions. Ablation of Spin4 in mice recapitulated the human phenotype with generalized overgrowth, including increased longitudinal bone growth. Growth plate analysis revealed increased cell proliferation in the proliferative zone and an increased number of progenitor chondrocytes in the resting zone. We also found evidence of decreased canonical Wnt signaling in growth plate chondrocytes, providing a potential explanation for the increased number of resting zone chondrocytes. Taken together, our findings provide strong evidence that SPIN4 is an epigenetic reader that negatively regulates mammalian body growth, and that loss of SPIN4 causes an overgrowth syndrome in humans, expanding our knowledge of the epigenetic regulation of human growth.

Authors

Julian C. Lui, Jacob Wagner, Elaine Zhou, Lijin Dong, Kevin M. Barnes, Youn Hee Jee, Jeffrey Baron

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