Genetics
Abstract
Hypochondroplasia (HCH) is a mild dwarfism caused by missense mutations in fibroblast growth factor receptor 3 (FGFR3), with the majority of cases resulting from a heterozygous p.Asn540Lys gain-of-function mutation. Here, we report the generation and characterization of the first mouse model (Fgfr3Asn534Lys/+) of HCH to our knowledge. Fgfr3Asn534Lys/+ mice exhibited progressive dwarfism and impairment of the synchondroses of the cranial base, resulting in defective formation of the foramen magnum. The appendicular and axial skeletons were both severely affected and we demonstrated an important role of FGFR3 in regulation of cortical and trabecular bone structure. Trabecular bone mineral density (BMD) of long bones and vertebral bodies was decreased, but cortical BMD increased with age in both tibiae and femurs. These results demonstrate that bones in Fgfr3Asn534Lys/+ mice, due to FGFR3 activation, exhibit some characteristics of osteoporosis. The present findings emphasize the detrimental effect of gain-of-function mutations in the Fgfr3 gene on long bone modeling during both developmental and aging processes, with potential implications for the management of elderly patients with hypochondroplasia and osteoporosis.
Authors
Léa Loisay, Davide Komla-Ebri, Anne Morice, Yann Heuzé, Camille Viaut, Amélie de La Seiglière, Nabil Kaci, Danny Chan, Audrey Lamouroux, Geneviève Baujat, J.H. Duncan Bassett, Graham R. Williams, Laurence Legeai-Mallet
Abstract
Radiation therapy is an effective cancer treatment although damages to healthy tissues are common. Here we analyzed cell-free, methylated DNA released from dying cells into the circulation to evaluate radiation-induced cellular damages in different tissues. To map the circulating DNA fragments to human and mouse tissues, we established sequencing-based, cell-type specific reference DNA methylation atlases. We found that cell-type specific DNA blocks were mostly hypomethylated and located within signature genes of cellular identity. Cell-free DNA fragments were captured from serum samples by hybridization to CpG-rich DNA panels and mapped to the DNA methylation atlases. In a mouse model, thoracic radiation-induced tissue damages were reflected by dose-dependent increases in lung endothelial and cardiomyocyte methylated DNA in serum. The analysis of serum samples from breast cancer patients undergoing radiation treatment revealed distinct dose-dependent and tissue-specific epithelial and endothelial responses to radiation across multiple organs. Strikingly, patients treated for right-sided breast cancers also showed increased hepatocyte and liver endothelial DNA in the circulation indicating the impact on liver tissues. Thus, changes in cell-free methylated DNA can uncover cell-type specific effects of radiation and provide a readout of the biologically effective radiation dose received by healthy tissues.
Authors
Megan E. McNamara, Netanel Loyfer, Amber J. Kiliti, Marcel O. Schmidt, Sapir Shabi-Porat, Sidharth S. Jain, Sarah Martinez Roth, A. Patrick McDeed IV, Nesreen Shahrour, Elizabeth Ballew, Yun-Tien Lin, Heng-Hong Li, Anne Deslattes Mays, Sonali Rudra, Anna T. Riegel, Keith Unger, Tommy Kaplan, Anton Wellstein
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
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
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
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
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
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
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
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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