Targeted alveolar regeneration with Frizzled-specific agonists.

Wnt ligands oligomerize Frizzled (Fzd) and Lrp5/6 receptors to control the specification and activity of stem cells in many species. How Wnt signaling is selectively activated in different stem cell populations, often within the same organ, is not understood. In lung alveoli, we show that distinct Wnt receptors are expressed by epithelial (Fzd5/6), endothelial (Fzd4), and stromal (Fzd1) cells. Fzd5 is uniquely required for alveolar epithelial stem cell activity, whereas fibroblasts utilize distinct Fzd receptors. Using an expanded repertoire of Fzd-Lrp agonists, we could activate canonical Wnt signaling in alveolar epithelial stem cells via either Fzd5 or, unexpectedly, non-canonical Fzd6. A Fzd5 agonist (Fzd5ag) or Fzd6ag stimulated alveolar epithelial stem cell activity and promoted survival in mice after lung injury, but only Fzd6ag promoted an alveolar fate in airway-derived progenitors. Therefore, we identify a potential strategy for promoting regeneration without exacerbating fibrosis during lung injury.

Near-chromosomal de novo assembly of Bengal tiger genome reveals genetic hallmarks of apex predation.

The tiger, a poster child for conservation, remains an endangered apex predator. Continued survival and recovery will require a comprehensive understanding of genetic diversity and the use of such information for population management. A high-quality tiger genome assembly will be an important tool for conservation genetics, especially for the Indian tiger, the most abundant subspecies in the wild. Here, we present high-quality near-chromosomal genome assemblies of a female and a male wild Indian tiger (Panthera tigris tigris). Our assemblies had a scaffold N50 of >140 Mb, with 19 scaffolds corresponding to the 19 numbered chromosomes, containing 95% of the genome. Our assemblies also enabled detection of longer stretches of runs of homozygosity compared to previous assemblies, which will help improve estimates of genomic inbreeding. Comprehensive genome annotation identified 26,068 protein-coding genes, including several gene families involved in key morphological features such as the teeth, claws, vision, olfaction, taste, and body stripes. We also identified 301 microRNAs, 365 small nucleolar RNAs, 632 transfer RNAs, and other noncoding RNA elements, several of which are predicted to regulate key biological pathways that likely contribute to the tiger's apex predatory traits. We identify signatures of positive selection in the tiger genome that are consistent with the Panthera lineage. Our high-quality genome will enable use of noninvasive samples for comprehensive assessment of genetic diversity, thus supporting effective conservation and management of wild tiger populations.

Genome-Wide Polygenic Score Predicts Large Number of High Risk Individuals in Monogenic Undiagnosed Young Onset Parkinson's Disease Patients from India.

Parkinson's disease (PD) is a genetically heterogeneous neurodegenerative disease with poorly defined environmental influences. Genomic studies of PD patients have identified disease-relevant monogenic genes, rare variants of significance, and polygenic risk-associated variants. In this study, whole genome sequencing data from 90 young onset Parkinson's disease (YOPD) individuals are analyzed for both monogenic and polygenic risk. The genetic variant analysis identifies pathogenic/likely pathogenic variants in eight of the 90 individuals (8.8%). It includes large homozygous coding exon deletions in PRKN and SNV/InDels in VPS13C, PLA2G6, PINK1, SYNJ1, and GCH1. Eleven rare heterozygous GBA coding variants are also identified in 13 (14.4%) individuals. In 34 (56.6%) individuals, one or more variants of uncertain significance (VUS) in PD/PD-relevant genes are observed. Though YOPD patients with a prioritized pathogenic variant show a low polygenic risk score (PRS), patients with prioritized VUS or no significant rare variants show an increased PRS odds ratio for PD. This study suggests that both significant rare variants and polygenic risk from common variants together may contribute to the genesis of PD. Further validation using a larger cohort of patients will confirm the interplay between monogenic and polygenic variants and their use in routine genetic PD diagnosis and risk assessment.

A renal cell carcinoma tumorgraft platform to advance precision medicine.

Renal cell carcinoma (RCC) encompasses a heterogenous group of tumors, but representative preclinical models are lacking. We previously showed that patient-derived tumorgraft (TG) models recapitulate the biology and treatment responsiveness. Through systematic orthotopic implantation of tumor samples from 926 ethnically diverse individuals into non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice, we generate a resource comprising 172 independently derived, stably engrafted TG lines from 148 individuals. TG lines are characterized histologically and genomically (whole-exome [n = 97] and RNA [n = 102] sequencing). The platform features a variety of histological and oncogenotypes, including TCGA clades further corroborated through orthogonal metabolomic analyses. We illustrate how it enables a deeper understanding of RCC biology; enables the development of tissue- and imaging-based molecular probes; and supports advances in drug development.

Determinants of renal cell carcinoma invasion and metastatic competence.

Metastasis is the principal cause of cancer related deaths. Tumor invasion is essential for metastatic spread. However, determinants of invasion are poorly understood. We addressed this knowledge gap by leveraging a unique attribute of kidney cancer. Renal tumors invade into large vessels forming tumor thrombi (TT) that migrate extending sometimes into the heart. Over a decade, we prospectively enrolled 83 ethnically-diverse patients undergoing surgical resection for grossly invasive tumors at UT Southwestern Kidney Cancer Program. In this study, we perform comprehensive histological analyses, integrate multi-region genomic studies, generate in vivo models, and execute functional studies to define tumor invasion and metastatic competence. We find that invasion is not always associated with the most aggressive clone. Driven by immediate early genes, invasion appears to be an opportunistic trait attained by subclones with diverse oncogenomic status in geospatial proximity to vasculature. We show that not all invasive tumors metastasize and identify determinants of metastatic competency. TT associated with metastases are characterized by higher grade, mTOR activation and a particular immune contexture. Moreover, TT grade is a better predictor of metastasis than overall tumor grade, which may have implications for clinical practice.

Retinoblastoma genetics screening and clinical management.

BACKGROUND: India accounts for 20% of the global retinoblastoma (RB) burden. However, the existing data on RB1 gene germline mutations and its influence on clinical decisions is minimally explored. METHODS: Fifty children with RB underwent complete clinical examination and appropriate multidisciplinary management. Screening of germline RB1 gene mutations was performed through next-generation sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis. The mutation and non-mutation groups were compared for clinical parameters especially severity, progression and recurrence. RESULTS: Twenty-nine patients had bilateral RB (BLRB) and 21 had unilateral RB (ULRB). The genetic analysis revealed 20 RB1 variations in 29 probands, inclusive of 3 novel mutations, known 16 mutations and heterozygous whole gene deletions. The mutation detection rate (MDR) was 86.2% in BLRB and 19% in ULRB. Associations of disease recurrence (p = 0.021), progression (p = 0.000) and higher percentage of optic nerve invasion, subretinal seeds and high-risk pathological factors were observed in the mutation group. Clinical management was influenced by the presence of germline mutations, particularly while deciding on enucleation, frequency of periodic follow up and radiotherapy. CONCLUSIONS: We identified novel RB1 mutations, and our mutation detection rate was on par with the previous global studies. In our study, genetic results influenced clinical management and we suggest that it should be an essential and integral component of RB-care in India and elsewhere.

A Norrin/Wnt surrogate antibody stimulates endothelial cell barrier function and rescues retinopathy.

The FZD4:LRP5:TSPAN12 receptor complex is activated by the secreted protein Norrin in retinal endothelial cells and leads to ßcatenin-dependent formation of the blood-retina-barrier during development and its homeostasis in adults. Mutations disrupting Norrin signaling have been identified in several congenital diseases leading to hypovascularization of the retina and blindness. Here, we developed F4L5.13, a tetravalent antibody designed to induce FZD4 and LRP5 proximity in such a way as to trigger ßcatenin signaling. Treatment of cultured endothelial cells with F4L5.13 rescued permeability induced by VEGF in part by promoting surface expression of junction proteins. Treatment of Tspan12-/- mice with F4L5.13 restored retinal angiogenesis and barrier function. F4L5.13 treatment also significantly normalized neovascularization in an oxygen-induced retinopathy model revealing a novel therapeutic strategy for diseases characterized by abnormal angiogenesis and/or barrier dysfunction.

Matrilineal analysis of mutations in the DMD gene in a multigenerational South Indian cohort using DMD gene panel sequencing.

BACKGROUND: Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder characterised by progressive irreversible muscle weakness, primarily of the skeletal and the cardiac muscles. DMD is characterised by mutations in the dystrophin gene, resulting in the absence or sparse quantities of dystrophin protein. A precise and timely molecular detection of DMD mutations encourages interventions such as carrier genetic counselling and in undertaking therapeutic measures for the DMD patients. RESULTS: In this study, we developed a 2.1 Mb custom DMD gene panel that spans the entire DMD gene, including the exons and introns. The panel also includes the probes against 80 additional genes known to be mutated in other muscular dystrophies. This custom DMD gene panel was used to identify single nucleotide variants (SNVs) and large deletions with precise breakpoints in 77 samples that included 24 DMD patients and their matrilineage across four generations. We used this panel to evaluate the inheritance pattern of DMD mutations in maternal subjects representing 24 DMD patients. CONCLUSION: Here we report our observations on the inheritance pattern of DMD gene mutations in matrilineage samples across four generations. Additionally, our data suggest that the DMD gene panel designed by us can be routinely used as a single genetic test to identify all DMD gene variants in DMD patients and the carrier mothers.

Human ACE2 receptor polymorphisms and altered susceptibility to SARS-CoV-2.

COVID-19 is a respiratory illness caused by a novel coronavirus called SARS-CoV-2. The viral spike (S) protein engages the human angiotensin-converting enzyme 2 (ACE2) receptor to invade host cells with ~10-15-fold higher affinity compared to SARS-CoV S-protein, making it highly infectious. Here, we assessed if ACE2 polymorphisms can alter host susceptibility to SARS-CoV-2 by affecting this interaction. We analyzed over 290,000 samples representing >400 population groups from public genomic datasets and identified multiple ACE2 protein-altering variants. Using reported structural data, we identified natural ACE2 variants that could potentially affect virus-host interaction and thereby alter host susceptibility. These include variants S19P, I21V, E23K, K26R, T27A, N64K, T92I, Q102P and H378R that were predicted to increase susceptibility, while variants K31R, N33I, H34R, E35K, E37K, D38V, Y50F, N51S, M62V, K68E, F72V, Y83H, G326E, G352V, D355N, Q388L and D509Y were predicted to be protective variants that show decreased binding to S-protein. Using biochemical assays, we confirmed that K31R and E37K had decreased affinity, and K26R and T92I variants showed increased affinity for S-protein when compared to wildtype ACE2. Consistent with this, soluble ACE2 K26R and T92I were more effective in blocking entry of S-protein pseudotyped virus suggesting that ACE2 variants can modulate susceptibility to SARS-CoV-2.

Anti-ferroptotic mechanism of IL4i1-mediated amino acid metabolism.

Interleukin-4-induced-1 (IL4i1) is an amino acid oxidase secreted from immune cells. Recent observations have suggested that IL4i1 is pro-tumorigenic via unknown mechanisms. As IL4i1 has homologs in snake venoms (L-amino acid oxidases [LAAO]), we used comparative approaches to gain insight into the mechanistic basis of how conserved amino acid oxidases regulate cell fate and function. Using mammalian expressed recombinant proteins, we found that venom LAAO kills cells via hydrogen peroxide generation. By contrast, mammalian IL4i1 is non-cytotoxic and instead elicits a cell protective gene expression program inhibiting ferroptotic redox death by generating indole-3-pyruvate (I3P) from tryptophan. I3P suppresses ferroptosis by direct free radical scavenging and through the activation of an anti-oxidative gene expression program. Thus, the pro-tumor effects of IL4i1 are likely mediated by local anti-ferroptotic pathways via aromatic amino acid metabolism, arguing that an IL4i1 inhibitor may modulate tumor cell death pathways.

Targeted editing of tomato carotenoid isomerase reveals the role of 5' UTR region in gene expression regulation.

KEY MESSAGE: A deletion created by CRISPR/Cas9 system in the 5' UTR of the carotenoid isomerase gene in tomato leads to downregulation of the gene resulting in the low conversion of prolycopene to lycopene. CRISPR/Cas9 based genome editing is an effective and useful tool adopted from the bacterial immune response system for altering specific, pre-determined DNA sequences in eukaryotes. Such targeted changes are finding wide application in human health as well as in precision breeding of crop plants for improved traits. Mutations in the coding and regulatory regions can have varying impacts on the function of the gene. In the current study, we demonstrate this on tomato carotenoid isomerase, a key gene in the carotenoid biosynthesis pathway. Mutations were generated in the 5' UTR and exon 1 of the carotenoid isomerase gene using CRISPR/Cas9 expression via Agrobacterium-mediated transformation of tomato variety Periyakulam 1 (PKM1). Molecular and biochemical studies demonstrate that CRISPR-mediated point mutations in the exon sequence lead to complete knockout of protein function whereas deletion in 5' UTR region lowers the expression of the gene leading to changes in plant phenotype.

Embryonic lethality and defective mammary gland development of activator-function impaired conditional knock-in Erbb3 V943R mice.

ERBB3 is a pseudokinase domain-containing member of the ERBB family of receptor tyrosine kinases (RTKs). Following ligand binding, ERBB receptors homo- or hetero-dimerize, leading to a head-to-tail arrangement of the intracellular kinase domains, where the "receiver" kinase domain of one ERBB is activated by the "activator" domain of the other ERBB in the dimer. In ERBB3, a conserved valine at codon 943 (V943) in the kinase C-terminal domain has been shown to be important for its function as an "activator" kinase in vitro. Here we report a knock-in mouse model where we have modified the endogenous Erbb3 allele to allow for tissue-specific conditional expression of Erbb3 V943R (Erbb3 CKI-V943R ). Additionally, we generated an Erbb3 D850N (Erbb3 CKI-D850N ) conditional knock-in mouse model where the conserved aspartate in the DFG motif of the pseudokinase domain was mutated to abolish any potential residual kinase activity. While Erbb3 D850N/D850N animals developed normally, homozygous Erbb3 V943R/V943R expression during development resulted in embryonic lethality. Further, tissue specific expression of Erbb3 V943R/V943R in the mammary gland epithelium following its activation using MMTV-Cre resulted in delayed elongation of the ductal network during puberty. Single-cell RNA-seq analysis of Erbb3 V943R/V943R mammary glands showed a reduction in a specific subset of fibrinogen-producing luminal epithelial cells.

Accurate assembly of the olive baboon (Papio anubis) genome using long-read and Hi-C data.

BACKGROUND: Baboons are a widely used nonhuman primate model for biomedical, evolutionary, and basic genetics research. Despite this importance, the genomic resources for baboons are limited. In particular, the current baboon reference genome Panu_3.0 is a highly fragmented, reference-guided (i.e., not fully de novo) assembly, and its poor quality inhibits our ability to conduct downstream genomic analyses. FINDINGS: Here we present a de novo genome assembly of the olive baboon (Papio anubis) that uses data from several recently developed single-molecule technologies. Our assembly, Panubis1.0, has an N50 contig size of ∼1.46 Mb (as opposed to 139 kb for Panu_3.0) and has single scaffolds that span each of the 20 autosomes and the X chromosome. CONCLUSIONS: We highlight multiple lines of evidence (including Bionano Genomics data, pedigree linkage information, and linkage disequilibrium data) suggesting that there are several large assembly errors in Panu_3.0, which have been corrected in Panubis1.0.

Phenotypic expression and clinical outcomes in a South Asian PRKAG2 cardiomyopathy cohort.

The PRKAG2 syndrome is a rare autosomal dominant phenocopy of sarcomeric hypertrophic cardiomyopathy (HCM), characterized by ventricular pre-excitation, progressive conduction system disease and left ventricular hypertrophy. This study describes the phenotype, genotype and clinical outcomes of a South-Asian PRKAG2 cardiomyopathy cohort over a 7-year period. Clinical, electrocardiographic, echocardiographic, and cardiac MRI data from 22 individuals with PRKAG2 variants (68% men; mean age 39.5 ± 18.1 years), identified at our HCM centre were studied prospectively. At initial evaluation, all of the patients were in NYHA functional class I or II. The maximum left ventricular wall thickness was 22.9 ± 8.7 mm and left ventricular ejection fraction was 53.4 ± 6.6%. Left ventricular hypertrophy was present in 19 individuals (86%) at baseline. 17 patients had an WPW pattern (77%). After a mean follow-up period of 7 years, 2 patients had undergone accessory pathway ablation, 8 patients (36%) underwent permanent pacemaker implantation (atrio-ventricular blocks-5; sinus node disease-2), 3 patients developed atrial fibrillation, 11 patients (50%) developed progressive worsening in NYHA functional class, and 6 patients (27%) experienced sudden cardiac death or equivalent. PRKAG2 cardiomyopathy must be considered in patients with HCM and progressive conduction system disease.

Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.

Proteogenomics analysis unveils a TFG-RET gene fusion and druggable targets in papillary thyroid carcinomas.

Papillary thyroid cancer (PTC) is the most common type of endocrine malignancy. By RNA-seq analysis, we identify a RET rearrangement in the tumour material of a patient who does not harbour any known RAS or BRAF mutations. This new gene fusion involves exons 1-4 from the 5' end of the Trk fused Gene (TFG) fused to the 3' end of RET tyrosine kinase leading to a TFG-RET fusion which transforms immortalized human thyroid cells in a kinase-dependent manner. TFG-RET oligomerises in a PB1 domain-dependent manner and oligomerisation of TFG-RET is required for oncogenic transformation. Quantitative proteomic analysis reveals the upregulation of E3 Ubiquitin ligase HUWE1 and DUBs like USP9X and UBP7 in both tumor and metastatic lesions, which is further confirmed in additional patients. Expression of TFG-RET leads to the upregulation of HUWE1 and inhibition of HUWE1 significantly reduces RET-mediated oncogenesis.

Restricted epitope specificity determined by variable region germline segment pairing in rodent antibody repertoires.

Antibodies from B-cell clonal lineages share sequence and structural properties as well as epitope specificity. Clonally unrelated antibodies can similarly share sequence and specificity properties and are said to be convergent. Convergent antibody responses against several antigens have been described in humans and mice and include different classes of shared sequence features. In particular, some antigens and epitopes can induce convergent responses of clonally unrelated antibodies with restricted heavy (VH) and light (VL) chain variable region germline segment usage without similarity in the heavy chain third complementarity-determining region (CDR H3), a critical specificity determinant. Whether these V germline segment-restricted responses reflect a general epitope specificity restriction of antibodies with shared VH/VL pairing is not known. Here, we investigated this question by determining patterns of antigen binding competition between clonally unrelated antigen-specific rat antibodies from paired-chain deep sequencing datasets selected based solely on VH/VL pairing. We found that antibodies with shared VH/VL germline segment pairings but divergent CDR H3 sequences almost invariably have restricted epitope specificity indicated by shared binding competition patterns. This epitope restriction included 82 of 85 clonally unrelated antibodies with 13 different VH/VL pairings binding in 8 epitope groups in 2 antigens. The corollary that antibodies with shared VH/VL pairing and epitope-restricted binding can accommodate widely divergent CDR H3 sequences was confirmed by in vitro selection of variants of anti-human epidermal growth factor receptor 2 antibodies known to mediate critical antigen interactions through CDR H3. Our results show that restricted epitope specificity determined by VH/VL germline segment pairing is a general property of rodent antigen-specific antibodies.

The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins.

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the 'venom-ome' and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 'venom-ome-specific toxins' (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery.

Exome sequencing reveals a novel splice site variant in HUWE1 gene in patients with suspected Say-Meyer syndrome.

Say-Meyer syndrome is a rare and clinically heterogeneous syndrome characterized by trigonocephaly, short stature, developmental delay and hypotelorism. Nine patients with this syndrome have been reported thus far although no causative gene has yet been identified. Here, we report two siblings with clinical phenotypes of Say-Meyer syndrome with moderate to severe intellectual disability and autism spectrum disorder. Cytogenetics and array-based comparative genomic hybridization did not reveal any chromosome abnormalities or copy number alterations. Exome sequencing of the patients revealed a novel X-linked recessive splice acceptor site variant c.145-2A > G in intron 5 of HUWE1 gene in both affected siblings. RT-PCR and sequencing revealed the use of an alternate cryptic splice acceptor site downstream, which led to deletion of six nucleotides resulting loss of two amino acids p.(Cys49-Glu50del) in HUWE1 protein. Deletion of these two amino acids, which are located in a highly conserved region, is predicted to be deleterious and quite likely to affect the function of HUWE1 protein. This is the first report of a potential candidate gene mutation for Say-Meyer syndrome, which was initially described four decades ago.