Ramipril therapy in integrin α1-null, autosomal recessive Alport mice triples lifespan: mechanistic clues from RNA-seq analysis.

The standard of care for patients with Alport syndrome (AS) is angiotensin-converting enzyme (ACE) inhibitors. In autosomal recessive Alport (ARAS) mice, ACE inhibitors double lifespan. We previously showed that deletion of Itga1 in Alport mice [double-knockout (DKO) mice] increased lifespan by 50%. This effect seemed dependent on the prevention of laminin 211-mediated podocyte injury. Here, we treated DKO mice with vehicle or ramipril starting at 4 weeks of age. Proteinuria and glomerular filtration rates were measured at 5-week intervals. Glomeruli were analyzed for laminin 211 deposition in the glomerular basement membrane (GBM) and GBM ultrastructure was analyzed using transmission electron microscopy (TEM). RNA sequencing (RNA-seq) was performed on isolated glomeruli at all time points and the results were compared with cultured podocytes overlaid (or not) with recombinant laminin 211. Glomerular filtration rate declined in ramipril-treated DKO mice between 30 and 35 weeks. Proteinuria followed these same patterns with normalization of foot process architecture in ramipril-treated DKO mice. RNA-seq revealed a decline in the expression of Foxc2, nephrin (Nphs1), and podocin (Nphs2) mRNAs, which was delayed in the ramipril-treated DKO mice. GBM accumulation of laminin 211 was delayed in ramipril-treated DKO mice, likely due to a role for α1ß1 integrin in CDC42 activation in Alport mesangial cells, which is required for mesangial filopodial invasion of the subendothelial spaces of the glomerular capillary loops. Ramipril synergized with Itga1 knockout, tripling lifespan compared with untreated ARAS mice. © 2023 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

NBSTRN Tools to Advance Newborn Screening Research and Support Newborn Screening Stakeholders.

Rapid advances in the screening, diagnosis, and treatment of genetic disorders have increased the number of conditions that can be detected through universal newborn screening (NBS). However, the addition of conditions to the Recommended Uniform Screening Panel (RUSP) and the implementation of nationwide screening has been a slow process taking several years to accomplish for individual conditions. Here, we describe web-based tools and resources developed and implemented by the newborn screening translational research network (NBSTRN) to advance newborn screening research and support NBS stakeholders worldwide. The NBSTRN's tools include the Longitudinal Pediatric Data Resource (LPDR), the NBS Condition Resource (NBS-CR), the NBS Virtual Repository (NBS-VR), and the Ethical, Legal, and Social Issues (ELSI) Advantage. Research programs, including the Inborn Errors of Metabolism Information System (IBEM-IS), BabySeq, EarlyCheck, and Family Narratives Use Cases, have utilized NBSTRN's tools and, in turn, contributed research data to further expand and refine these resources. Additionally, we discuss ongoing tool development to facilitate the expansion of genetic disease screening in increasingly diverse populations. In conclusion, NBSTRN's tools and resources provide a trusted platform to enable NBS stakeholders to advance NBS research and improve clinical care for patients and their families.

Functional variants identify sex-specific genes and pathways in Alzheimer's Disease.

The incidence of Alzheimer's Disease in females is almost double that of males. To search for sex-specific gene associations, we build a machine learning approach focused on functionally impactful coding variants. This method can detect differences between sequenced cases and controls in small cohorts. In the Alzheimer's Disease Sequencing Project with mixed sexes, this approach identified genes enriched for immune response pathways. After sex-separation, genes become specifically enriched for stress-response pathways in male and cell-cycle pathways in female. These genes improve disease risk prediction in silico and modulate Drosophila neurodegeneration in vivo. Thus, a general approach for machine learning on functionally impactful variants can uncover sex-specific candidates towards diagnostic biomarkers and therapeutic targets.

Epigenetic age acceleration among survivors of pediatric medulloblastoma and primitive neuroectodermal tumor.

Survivors of childhood central nervous system (CNS) tumors experience early-onset aging-related phenotypes. DNA methylation (DNAm) age is an emerging epigenetic biomarker of physiologic age and may be predictive of chronic health conditions in long-term survivors. This report describes the course of epigenetic age acceleration using post-diagnosis blood samples (median: 3.9 years post-diagnosis; range: 0.04-15.96) from 83 survivors of pediatric CNS tumors. Epigenetic age acceleration was detected in 72% of patients, with an average difference between chronologic and DNAm age of 2.58 years (95% CI: 1.75-3.41, p < 0.001). Time from diagnosis to sample collection correlated with the magnitude of epigenetic age acceleration.

Using Long-Term Follow-Up Data to Classify Genetic Variants in Newborn Screened Conditions.

With the rapid increase in publicly available sequencing data, healthcare professionals are tasked with understanding how genetic variation informs diagnosis and affects patient health outcomes. Understanding the impact of a genetic variant in disease could be used to predict susceptibility/protection and to help build a personalized medicine profile. In the United States, over 3.8 million newborns are screened for several rare genetic diseases each year, and the follow-up testing of screen-positive newborns often involves sequencing and the identification of variants. This presents the opportunity to use longitudinal health information from these newborns to inform the impact of variants identified in the course of diagnosis. To test this, we performed secondary analysis of a 10-year natural history study of individuals diagnosed with metabolic disorders included in newborn screening (NBS). We found 564 genetic variants with accompanying phenotypic data and identified that 161 of the 564 variants (29%) were not included in ClinVar. We were able to classify 139 of the 161 variants (86%) as pathogenic or likely pathogenic. This work demonstrates that secondary analysis of longitudinal data collected as part of NBS finds unreported genetic variants and the accompanying clinical information can inform the relationship between genotype and phenotype.

Glomerular basement membrane deposition of collagen α1(III) in Alport glomeruli by mesangial filopodia injures podocytes via aberrant signaling through DDR1 and integrin α2ß1.

In Alport mice, activation of the endothelin A receptor (ETA R) in mesangial cells results in sub-endothelial invasion of glomerular capillaries by mesangial filopodia. Filopodia deposit mesangial matrix in the glomerular basement membrane (GBM), including laminin 211 which activates NF-κB, resulting in induction of inflammatory cytokines. Herein we show that collagen α1(III) is also deposited in the GBM. Collagen α1(III) localized to the mesangium in wild-type mice and was found in both the mesangium and the GBM in Alport mice. We show that collagen α1(III) activates discoidin domain receptor family, member 1 (DDR1) receptors both in vitro and in vivo. To elucidate whether collagen α1(III) might cause podocyte injury, cultured murine Alport podocytes were overlaid with recombinant collagen α1(III), or not, for 24 h and RNA was analyzed by RNA sequencing (RNA-seq). These same cells were subjected to siRNA knockdown for integrin α2 or DDR1 and the RNA was analyzed by RNA-seq. Results were validated in vivo using RNA-seq from RNA isolated from wild-type and Alport mouse glomeruli. Numerous genes associated with podocyte injury were up- or down-regulated in both Alport glomeruli and cultured podocytes treated with collagen α1(III), 18 of which have been associated previously with podocyte injury or glomerulonephritis. The data indicate α2ß1 integrin/DDR1 co-receptor signaling as the dominant regulatory mechanism. This may explain earlier studies where deletion of either DDR1 or α2ß1 integrin in Alport mice ameliorates renal pathology. © 2022 Boys Town National Research Hospital. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genome interpretation using in silico predictors of variant impact.

Estimating the effects of variants found in disease driver genes opens the door to personalized therapeutic opportunities. Clinical associations and laboratory experiments can only characterize a tiny fraction of all the available variants, leaving the majority as variants of unknown significance (VUS). In silico methods bridge this gap by providing instant estimates on a large scale, most often based on the numerous genetic differences between species. Despite concerns that these methods may lack reliability in individual subjects, their numerous practical applications over cohorts suggest they are already helpful and have a role to play in genome interpretation when used at the proper scale and context. In this review, we aim to gain insights into the training and validation of these variant effect predicting methods and illustrate representative types of experimental and clinical applications. Objective performance assessments using various datasets that are not yet published indicate the strengths and limitations of each method. These show that cautious use of in silico variant impact predictors is essential for addressing genome interpretation challenges.

RNA-seq analysis of gene expression profiles in isolated stria vascularis from wild-type and Alport mice reveals key pathways underling Alport strial pathogenesis.

Previous work demonstrates that the hearing loss in Alport mice is caused by defects in the stria vascularis. As the animals age, progressive thickening of strial capillary basement membranes (SCBMs) occurs associated with elevated levels of extracellular matrix expression and hypoxia-related gene and protein expression. These conditions render the animals susceptible to noise-induced hearing loss. In an effort to develop a more comprehensive understanding of how the underlying mutation in the COL4A3 gene influences homeostasis in the stria vascularis, we performed vascular permeability studies combined with RNA-seq analysis using isolated stria vascularis from 7-week old wild-type and Alport mice on the 129 Sv background. Alport SCBMs were found to be less permeable than wild-type littermates. RNA-seq and bioinformatics analysis revealed 68 genes were induced and 61 genes suppressed in the stria from Alport mice relative to wild-type using a cut-off of 2-fold. These included pathways involving transcription factors associated with the regulation of pro-inflammatory responses as well as cytokines, chemokines, and chemokine receptors that are up- or down-regulated. Canonical pathways included modulation of genes associated with glucose and glucose-1-PO4 degradation, NAD biosynthesis, histidine degradation, calcium signaling, and glutamate receptor signaling (among others). In all, the data point to the Alport stria being in an inflammatory state with disruption in numerous metabolic pathways indicative of metabolic stress, a likely cause for the susceptibility of Alport mice to noise-induced hearing loss under conditions that do not cause permanent hearing loss in age/strain-matched wild-type mice. The work lays the foundation for studies aimed at understanding the nature of strial pathology in Alport mice. The modulation of these genes under conditions of therapeutic intervention may provide important pre-clinical data to justify trials in humans afflicted with the disease.