Genomics Publications

Varela APM, Sant'Anna FH, Dos Santos AV, Prichula J, Comerlato J, Dos Santos GT, Wendland E. Genomic evidence of SARS-CoV-2 reinfection cases in southern Brazil. Arch Virol 2023;168(1):19.Abstract
Cases of reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been reported worldwide. We investigated reinfection cases in a set of more than 30,000 samples, and the SARS-CoV-2 genomes from selected samples from four patients with at least two positive diagnoses with an interval ≥ 45 days between tests were sequenced and analyzed. Comparative genomic and phylogenetic analysis confirmed three reinfection cases and suggested that the fourth one was caused by a virus of the same lineage. Viral sequencing is crucial for understanding the natural course of reinfections and for planning public health strategies for management of COVID-19.
Pons S, Frapy E, Sereme Y, Gaultier C, Lebreton F, Kropec A, Danilchanka O, Schlemmer L, Schrimpf C, Allain M, Angoulvant F, Lecuyer H, Bonacorsi S, Aschard H, Sokol H, Cywes-Bentley C, Mekalanos JJ, Guillard T, Pier GB, Roux D, Skurnik D. A high-throughput sequencing approach identifies immunotherapeutic targets for bacterial meningitis in neonates. EBioMedicine 2023;88:104439.Abstract
BACKGROUND: Worldwide, Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but full understanding of the pathogenesis of this disease is not yet achieved. Moreover, to date, no vaccine is available against bacterial neonatal meningitis. METHODS: Here, we used Transposon Sequencing of saturated banks of mutants (TnSeq) to evaluate E. coli K1 genetic fitness in murine neonatal meningitis. We identified E. coli K1 genes encoding for factors important for systemic dissemination and brain infection, and focused on products with a likely outer-membrane or extra-cellular localization, as these are potential vaccine candidates. We used in vitro and in vivo models to study the efficacy of active and passive immunization. RESULTS: We selected for further study the conserved surface polysaccharide Poly-β-(1-6)-N-Acetyl Glucosamine (PNAG), as a strong candidate for vaccine development. We found that PNAG was a virulence factor in our animal model. We showed that both passive and active immunization successfully prevented and/or treated meningitis caused by E. coli K1 in neonatal mice. We found an excellent opsonophagocytic killing activity of the antibodies to PNAG and in vitro these antibodies were also able to decrease binding, invasion and crossing of E. coli K1 through two blood brain barrier cell lines. Finally, to reinforce the potential of PNAG as a vaccine candidate in bacterial neonatal meningitis, we demonstrated that Group B Streptococcus, the main cause of neonatal meningitis in developed countries, also produced PNAG and that antibodies to PNAG could protect in vitro and in vivo against this major neonatal pathogen. INTERPRETATION: Altogether, these results indicate the utility of a high-throughput DNA sequencing method to identify potential immunotherapy targets for a pathogen, including in this study a potential broad-spectrum target for prevention of neonatal bacterial infections. FUNDINGS: ANR Seq-N-Vaq, Charles Hood Foundation, Hearst Foundation, and Groupe Pasteur Mutualité.
Liu C-H, Yemanyi F, Bora K, Kushwah N, Blomfield AK, Kamenecka TM, SanGiovanni JP, Sun Y, Solt LA, Chen J. Genetic deficiency and pharmacological modulation of RORα regulate laser-induced choroidal neovascularization. Aging (Albany NY) 2023;15(1):37-52.Abstract
Choroidal neovascularization (CNV) causes acute vision loss in neovascular age-related macular degeneration (AMD). Genetic variations of the nuclear receptor RAR-related orphan receptor alpha (RORα) have been linked with neovascular AMD, yet its specific role in pathological CNV development is not entirely clear. In this study, we showed that Rora was highly expressed in the mouse choroid compared with the retina, and genetic loss of RORα in Staggerer mice (Rorasg/sg) led to increased expression levels of Vegfr2 and Tnfa in the choroid and retinal pigment epithelium (RPE) complex. In a mouse model of laser-induced CNV, RORα expression was highly increased in the choroidal/RPE complex post-laser, and loss of RORα in Rorasg/sg eyes significantly worsened CNV with increased lesion size and vascular leakage, associated with increased levels of VEGFR2 and TNFα proteins. Pharmacological inhibition of RORα also worsened CNV. In addition, both genetic deficiency and inhibition of RORα substantially increased vascular growth in isolated mouse choroidal explants ex vivo. RORα inhibition also promoted angiogenic function of human choroidal endothelial cell culture. Together, our results suggest that RORα negatively regulates pathological CNV development in part by modulating angiogenic response of the choroidal endothelium and inflammatory environment in the choroid/RPE complex.
Wu W, Ma G, Qi H, Dong L, Chen F, Wang Y, Mao X, Guo X, Cui J, Matsubara JA, Vanhaesebroeck B, Yan X, Zhao G, Zhang S, Lei H. Genome Editing of Pik3cd Impedes Abnormal Retinal Angiogenesis. Hum Gene Ther 2023;34(1-2):30-41.Abstract
Abnormal angiogenesis is associated with myriad human diseases, including proliferative diabetic retinopathy (PDR). Signaling transduction through phosphoinositide 3-kinases (PI3Ks) plays a critical role in angiogenesis. Herein, we showed that p110δ, the catalytic subunit of PI3Kδ, was highly expressed in pathological retinal vascular endothelial cells (ECs) in a mouse model of oxygen-induced retinopathy (OIR) and in fibrovascular membranes from patients with PDR. To explore novel intervention with PI3Kδ expression, we developed a recombinant dual adeno-associated viral (rAAV) system for delivering CRISPR/Cas9 in which Streptococcus pyogenes (Sp) Cas9 expression was driven by an endothelial specific promoter of the intercellular adhesion molecule 2 (pICAM2) to edit genomic Pik3cd, the gene encoding p110δ. We then demonstrated that infection of cultured mouse vascular ECs with the dual rAAV1s of rAAV1-pICAM2-SpCas9 and rAAV1-SpGuide targeting genomic Pik3cd resulted in 80% DNA insertion/deletion in the locus of genomic Pik3cd and 70% depletion of p110δ expression. Furthermore, we showed that in the mouse model of OIR editing retinal Pik3cd with the dual rAAV1s resulted in not only a significant decrease in p110δ expression, and Akt activation, but also a dramatic reduction in pathological retinal angiogenesis. These findings reveal that Pik3cd editing is a novel approach to treating abnormal retinal angiogenesis.
Nakamichi K, Akileswaran L, Meirick T, Lee MD, Chodosh J, Rajaiya J, Stroman D, Wolf-Yadlin A, Jackson Q, Holtz BW, Lee AY, Lee CS, Van Gelder RN, Van Gelder RN. Machine Learning Prediction of Adenovirus D8 Conjunctivitis Complications from Viral Whole-Genome Sequence. Ophthalmol Sci 2022;2(4):100166.Abstract
OBJECTIVE: To obtain complete DNA sequences of adenoviral (AdV) D8 genome from patients with conjunctivitis and determine the relation of sequence variation to clinical outcomes. DESIGN: This study is a post hoc analysis of banked conjunctival swab samples from the BAYnovation Study, a previously conducted, randomized controlled clinical trial for AdV conjunctivitis. PARTICIPANTS: Ninety-six patients with AdV D8-positive conjunctivitis who received placebo treatment in the BAYnovation Study were included in the study. METHODS: DNA from conjunctival swabs was purified and subjected to whole-genome viral DNA sequencing. Adenovirus D8 variants were identified and correlated with clinical outcomes, including 2 machine learning methods. MAIN OUTCOME MEASURES: Viral DNA sequence and development of subepithelial infiltrates (SEIs) were the main outcome measures. RESULTS: From initial sequencing of 80 AdV D8-positive samples, full adenoviral genome reconstructions were obtained for 71. A total of 630 single-nucleotide variants were identified, including 156 missense mutations. Sequence clustering revealed 3 previously unappreciated viral clades within the AdV D8 type. The likelihood of SEI development differed significantly between clades, ranging from 83% for Clade 1 to 46% for Clade 3. Genome-wide analysis of viral single-nucleotide polymorphisms failed to identify single-gene determinants of outcome. Two machine learning models were independently trained to predict clinical outcome using polymorphic sequences. Both machine learning models correctly predicted development of SEI outcomes in a newly sequenced validation set of 16 cases (P = 1.5 × 10-5). Prediction was dependent on ensemble groups of polymorphisms across multiple genes. CONCLUSIONS: Adenovirus D8 has ≥ 3 prevalent molecular substrains, which differ in propensity to result in SEIs. Development of SEIs can be accurately predicted from knowledge of full viral sequence. These results suggest that development of SEIs in AdV D8 conjunctivitis is largely attributable to pathologic viral sequence variants within the D8 type and establishes machine learning paradigms as a powerful technique for understanding viral pathogenicity.
Sangermano R, Biswas P, Sullivan LS, Place EM, Borooah S, Straubhaar J, Pierce EA, Daiger SP, Bujakowska KM, Ayaggari R. Identification of a novel large multigene deletion and a frameshift indel in PDE6B as the underlying cause of early-onset recessive rod-cone degeneration. Cold Spring Harb Mol Case Stud 2022;8(7)Abstract
A family, with two affected identical twins with early-onset recessive inherited retinal degeneration, was analyzed to determine the underlying genetic cause of pathology. Exome sequencing revealed a rare and previously reported causative variant (c.1923_1969delinsTCTGGG; p.Asn643Glyfs*29) in the PDE6B gene in the affected twins and their unaffected father. Further investigation, using genome sequencing, identified a novel ∼7.5-kb deletion (Chr 4:670,405-677,862del) encompassing the ATP5ME gene, part of the 5' UTR of MYL5, and a 378-bp (Chr 4:670,405-670,782) region from the 3' UTR of PDE6B in the affected twins and their unaffected mother. Both variants segregated with disease in the family. Analysis of the relative expression of PDE6B, in peripheral blood cells, also revealed a significantly lower level of PDE6B transcript in affected siblings compared to a normal control. PDE6B is associated with recessive rod-cone degeneration and autosomal dominant congenital stationary night blindness. Ophthalmic evaluation of these patients showed night blindness, fundus abnormalities, and peripheral vision loss, which are consistent with PDE6B-associated recessive retinal degeneration. These findings suggest that the loss of PDE6B transcript resulting from the compound heterozygous pathogenic variants is the underlying cause of recessive rod-cone degeneration in the study family.
Kishi JY, Liu N, West ER, Sheng K, Jordanides JJ, Serrata M, Cepko CL, Saka SK, Yin P. Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing. Nat Methods 2022;19(11):1393-1402.Abstract
We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.
Zabaleta N, Bhatt U, Hérate C, Maisonnasse P, Sanmiguel J, Diop C, Castore S, Estelien R, Li D, Dereuddre-Bosquet N, Cavarelli M, Gallouët A-S, Pascal Q, Naninck T, Kahlaoui N, Lemaitre J, Relouzat F, Ronzitti G, Thibaut HJ, Montomoli E, Wilson JM, Le Grand R, Vandenberghe LH. Durable immunogenicity, adaptation to emerging variants, and low-dose efficacy of an AAV-based COVID-19 vaccine platform in macaques. Mol Ther 2022;30(9):2952-2967.Abstract
The COVID-19 pandemic continues to have devastating consequences on health and economy, even after the approval of safe and effective vaccines. Waning immunity, the emergence of variants of concern, breakthrough infections, and lack of global vaccine access and acceptance perpetuate the epidemic. Here, we demonstrate that a single injection of an adenoassociated virus (AAV)-based COVID-19 vaccine elicits at least 17-month-long neutralizing antibody responses in non-human primates at levels that were previously shown to protect from viral challenge. To improve the scalability of this durable vaccine candidate, we further optimized the vector design for greater potency at a reduced dose in mice and non-human primates. Finally, we show that the platform can be rapidly adapted to other variants of concern to robustly maintain immunogenicity and protect from challenge. In summary, we demonstrate this class of AAV can provide durable immunogenicity, provide protection at dose that is low and scalable, and be adapted readily to novel emerging vaccine antigens thus may provide a potent tool in the ongoing fight against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).
Guindolet D, Woodward AM, Gabison EE, Argüeso P. Glycogene Expression Profile of Human Limbal Epithelial Cells with Distinct Clonogenic Potential. Cells 2022;11(9)Abstract
Glycans function as valuable markers of stem cells but also regulate the ability of these cells to self-renew and differentiate. Approximately 2% of the human genome encodes for proteins that are involved in the biosynthesis and recognition of glycans. In the present study, we evaluated the expression of a small subset of glycogenes in human limbal epithelial cells with distinct clonogenic potential. Individual clones were classified as abortive or clonogenic, based on the fraction of the terminal colonies produced; clones leading exclusively to terminal colonies were referred to as abortive while those with half or fewer terminal colonies were referred to as clonogenic. An analysis of glycogene expression in clonogenic cultures revealed a high content of transcripts regulating the galactose and mannose metabolic pathways. Abortive clones were characterized by increased levels of GCNT4 and FUCA2, genes that are responsible for the branching of mucin-type O-glycans and the hydrolysis of fucose residues on N-glycans, respectively. The expansion of primary cultures of human limbal epithelial cells for 10 days resulted in stratification and a concomitant increase in MUC16, GCNT4 and FUCA2 expression. These data indicate that the clonogenic potential of human limbal epithelial cells is associated with specific glycosylation pathways. Mucin-type O-glycan branching and increased fucose metabolism are linked to limbal epithelial cell differentiation.
Eraslan G, Drokhlyansky E, Anand S, Fiskin E, Subramanian A, Slyper M, Wang J, Van Wittenberghe N, Rouhana JM, Waldman J, Ashenberg O, Lek M, Dionne D, Win TS, Cuoco MS, Kuksenko O, Tsankov AM, Branton PA, Marshall JL, Greka A, Getz G, Segrè AV, Aguet F, Rozenblatt-Rosen O, Ardlie KG, Regev A. Single-nucleus cross-tissue molecular reference maps toward understanding disease gene function. Science 2022;376(6594):eabl4290.Abstract
Understanding gene function and regulation in homeostasis and disease requires knowledge of the cellular and tissue contexts in which genes are expressed. Here, we applied four single-nucleus RNA sequencing methods to eight diverse, archived, frozen tissue types from 16 donors and 25 samples, generating a cross-tissue atlas of 209,126 nuclei profiles, which we integrated across tissues, donors, and laboratory methods with a conditional variational autoencoder. Using the resulting cross-tissue atlas, we highlight shared and tissue-specific features of tissue-resident cell populations; identify cell types that might contribute to neuromuscular, metabolic, and immune components of monogenic diseases and the biological processes involved in their pathology; and determine cell types and gene modules that might underlie disease mechanisms for complex traits analyzed by genome-wide association studies.
Zhang C, Owen LA, Lillvis JH, Zhang SX, Kim IK, Deangelis MM. AMD Genomics: Non-Coding RNAs as Biomarkers and Therapeutic Targets. J Clin Med 2022;11(6)Abstract
Age-related macular degeneration (AMD) is a progressive neurodegenerative disease that is the world's leading cause of blindness in the aging population. Although the clinical stages and forms of AMD have been elucidated, more specific prognostic tools are required to determine when patients with early and intermediate AMD will progress into the advanced stages of AMD. Another challenge in the field has been the appropriate development of therapies for intermediate AMD and advanced atrophic AMD. After numerous negative clinical trials, an anti-C5 agent and anti-C3 agent have recently shown promising results in phase 3 clinical trials, in terms of slowing the growth of geographic atrophy, an advanced form of AMD. Interestingly, both drugs appear to be associated with an increased incidence of wet AMD, another advanced form of the disease, and will require frequent intravitreal injections. Certainly, there remains a need for other therapeutic agents with the potential to prevent progression to advanced stages of the disease. Investigation of the role and clinical utility of non-coding RNAs (ncRNAs) is a major advancement in biology that has only been minimally applied to AMD. In the following review, we discuss the clinical relevance of ncRNAs in AMD as both biomarkers and therapeutic targets.
Simcoe MJ, Shah A, Fan BJ, Choquet H, Weisschuh N, Waseem NH, Jiang C, Melles RB, Ritch R, Mahroo OA, Wissinger B, Jorgenson E, Wiggs JL, Garway-Heath DF, Hysi PG, Hammond CJ. Genome-Wide Association Study Identifies Two Common Loci Associated with Pigment Dispersion Syndrome/Pigmentary Glaucoma and Implicates Myopia in its Development. Ophthalmology 2022;129(6):626-636.Abstract
PURPOSE: To identify genetic variants associated with pigment dispersion syndrome (PDS) and pigmentary glaucoma (PG) in unrelated patients and to further understand the genetic and potentially causal relationships between PDS and associated risk factors. DESIGN: A 2-stage genome-wide association meta-analysis with replication and subsequent in silico analyses including Mendelian randomization. PARTICIPANTS: A total of 574 cases with PG or PDS and 52 627 controls of European descent. METHODS: Genome-wide association analyses were performed in 4 cohorts and meta-analyzed in 3 stages: (1) a discovery meta-analysis was performed in 3 cohorts, (2) replication was performed in the fourth cohort, and (3) all 4 cohorts were meta-analyzed to increase statistical power. Two-sample Mendelian randomization was used to determine whether refractive error and intraocular pressure exert causal effects over PDS. MAIN OUTCOME MEASURES: The association of genetic variants with PDS and whether myopia exerts causal effects over PDS. RESULTS: Significant association was present at 2 novel loci for PDS/PG. These loci and follow-up analyses implicate the genes gamma secretase activator protein (GSAP) (lead single nucleotide polymorphism [SNP]: rs9641220, P = 6.0×10-10) and glutamate metabotropic receptor 5 (GRM5)/TYR (lead SNP: rs661177, P = 3.9×10-9) as important factors in disease risk. Mendelian randomization showed significant evidence that negative refractive error (myopia) exerts a direct causal effect over PDS (P = 8.86×10-7). CONCLUSIONS: Common SNPs relating to the GSAP and GRM5/TYR genes are associated risk factors for the development of PDS and PG. Although myopia is a known risk factor, this study uses genetic data to demonstrate that myopia is, in part, a cause of PDS and PG.
Catomeris AJ, Ballios BG, Sangermano R, Wagner NE, Comander JI, Pierce EA, Place EM, Bujakowska KM, Huckfeldt RM. Novel RCBTB1 variants causing later-onset non-syndromic retinal dystrophy with macular chorioretinal atrophy. Ophthalmic Genet 2022;43(3):332-339.Abstract
BACKGROUND: Variants in RCBTB1 were recently described to cause a retinal dystrophy with only eight families described to date and a predominant phenotype of macular atrophy and peripheral reticular degeneration. Here, we further evaluate the genotypic and phenotypic characteristics of biallelic RCBTB1-associated retinal dystrophy in a North American clinic population. METHODS: A retrospective analysis of genetic and clinical features was performed in individuals with biallelic variants in RCBTB1. RESULTS: Three unrelated individuals of French-Canadian descent with rare biallelic RCBTB1 variants were identified. All individuals shared a novel p.(Ser342Leu) missense variant; one patient was homozygous whereas the other two each possessed a second unique novel variant p.(Gln120*) and p.(Pro224Leu). All three had macula-predominant disease with symptom onset in the fifth decade of life. CONCLUSION: This report adds to the genetic diversity of RCBTB1-associated disease. These cases confirm the later-onset, relative to many other retinal dystrophies, and macular focus of disease described in most cases to-date. They are thus a reminder of considering hereditary disease in the differential for later-onset macular atrophy.
Natera-de Benito D, Jurgens JA, Yeung A, Zaharieva IT, Manzur A, DiTroia SP, Di Gioia SA, Pais L, Pini V, Barry BJ, Chan W-M, Elder JE, Christodoulou J, Hay E, England EM, Munot P, Hunter DG, Feng L, Ledoux D, O'Donnell-Luria A, Phadke R, Engle EC, Sarkozy A, Muntoni F. Recessive variants in COL25A1 gene as novel cause of arthrogryposis multiplex congenita with ocular congenital cranial dysinnervation disorder. Hum Mutat 2022;43(4):487-498.Abstract
A proper interaction between muscle-derived collagen XXV and its motor neuron-derived receptors protein tyrosine phosphatases σ and δ (PTP σ/δ) is indispensable for intramuscular motor innervation. Despite this, thus far, pathogenic recessive variants in the COL25A1 gene had only been detected in a few patients with isolated ocular congenital cranial dysinnervation disorders. Here we describe five patients from three unrelated families with recessive missense and splice site COL25A1 variants presenting with a recognizable phenotype characterized by arthrogryposis multiplex congenita with or without an ocular congenital cranial dysinnervation disorder phenotype. The clinical features of the older patients remained stable over time, without central nervous system involvement. This study extends the phenotypic and genotypic spectrum of COL25A1 related conditions, and further adds to our knowledge of the complex process of intramuscular motor innervation. Our observations indicate a role for collagen XXV in regulating the appropriate innervation not only of extraocular muscles, but also of bulbar, axial, and limb muscles in the human.
Fote GM, Geller NR, Efstathiou NE, Hendricks N, Vavvas DG, Reidling JC, Thompson LM, Steffan JS. Isoform-dependent lysosomal degradation and internalization of apolipoprotein E requires autophagy proteins. J Cell Sci 2022;135(2)Abstract
The human apolipoprotein E4 isoform (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and lysosomal dysfunction has been implicated in AD pathogenesis. We found, by examining cells stably expressing each APOE isoform, that APOE4 increases lysosomal trafficking, accumulates in enlarged lysosomes and late endosomes, alters autophagic flux and the abundance of autophagy proteins and lipid droplets, and alters the proteomic contents of lysosomes following internalization. We investigated APOE-related lysosomal trafficking further in cell culture, and found that APOE from the post-Golgi compartment is degraded through autophagy. We found that this autophagic process requires the lysosomal membrane protein LAMP2 in immortalized neuron-like and hepatic cells, and in mouse brain tissue. Several macroautophagy-associated proteins were also required for autophagic degradation and internalization of APOE in hepatic cells. The dysregulated autophagic flux and lysosomal trafficking of APOE4 that we observed suggest a possible novel mechanism that might contribute to AD pathogenesis. This article has an associated First Person interview with the first author of the paper.