Biology subjects

Fresard, L.

Publications and source records attributed to Fresard, L..

7 recordsLinked to original sources

bioRxiv2023

Systematically testing human HMBS missense variants to reveal mechanism and pathogenic variation

Defects in hydroxymethylbilane synthase (HMBS) can cause Acute Intermittent Porphyria (AIP), an acute neurological disease. Although sequencing-based diagnosis can be definitive, ~[1/3] of clinical HMBS variants are missense variants, and most clinically-reported HMBS missense variants are designated as "variants of uncertain significance" (VUS). Using saturation mutagenesis, en masse selection, and sequencing, we applied a multiplexed validated assay to both the erythroid-specific and ubiquitous isoforms of HMBS, obtaining confident functional impact scores for >84% of all possible amino-acid substitutions. The resulting variant effect maps generally agreed with biochemical expectation. However, the maps showed variants at the dimerization interface to be unexpectedly well tolerated, and suggested residue roles in active site dynamics that were supported by molecular dynamics simulations. Most importantly, these HMBS variant effect maps can help discriminate pathogenic from benign variants, proactively providing evidence even for yet-to-be-observed clinical missense variants.

molecular biology↗

bioRxiv2022

An algorithm to detect abnormal mRNA splicing and assess its clinical impact in individuals undergoing genetic testing for hereditary cancer syndromes

Nearly 14% of disease-causing germline variants result from disruption of mRNA splicing. Most (67%) DNA variants predicted in silico to disrupt splicing end up classified as variants of uncertain significance (VUS). We developed and validated an analytic workflow -- Splice Effect Event Resolver (SPEER) -- that uses mRNA sequencing to reveal significant deviations in splicing, pinpoints the DNA variants potentially responsible, and measures the deleterious effect of the altered splicing on mRNA transcripts, providing evidence to assess the pathogenicity of the variant. SPEER was used to analyze leukocyte RNA encoding 63 hereditary cancer syndrome genes in 20,317 individuals undergoing clinical genetic testing. Among 3,563 (17.5%) individuals with at least one DNA variant predicted to affect splicing, 971 (4.8%) had altered splicing with a deleterious effect on the transcript and 31 had altered splicing due to a DNA variant located outside our laboratorys reportable range. Integrating SPEER results into variant interpretation allowed reclassification of VUS to P/LP in 0.4% and to B/LB in 5.9% of the 20,317 patients. SPEER evidence had a significantly higher impact on allowing P/LP and B/LB interpretations in non-White individuals than in non-Hispanic White individuals, illustrating that evidence derived from RNA splicing analysis may reduce ethnic/ancestral disparities in genetic testing.

genetics↗

bioRxiv2022

Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration

In aging, skeletal muscle strength and regenerative capacity declines due, in part, to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here we capitalize on mass-cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blocking of CD47 alternative polyadenylation or antibody blockade of CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.

cell biology↗

bioRxiv2020

Single-cell epigenomic identification of inherited risk loci in Alzheimer's and Parkinson's disease

Genome-wide association studies (GWAS) have identified thousands of variants associated with disease phenotypes. However, the majority of these variants do not alter coding sequences, making it difficult to assign their function. To this end, we present a multi-omic epigenetic atlas of the adult human brain through profiling of the chromatin accessibility landscapes and three-dimensional chromatin interactions of seven brain regions across a cohort of 39 cognitively healthy individuals. Single-cell chromatin accessibility profiling of 70,631 cells from six of these brain regions identifies 24 distinct cell clusters and 359,022 cell type-specific regulatory elements, capturing the regulatory diversity of the adult brain. We develop a machine learning classifier to integrate this multi-omic framework and predict dozens of functional single nucleotide polymorphisms (SNPs), nominating gene and cellular targets for previously orphaned GWAS loci. These predictions both inform well-studied disease-relevant genes, such as BIN1 in microglia for Alzheimers disease (AD) and reveal novel gene-disease associations, such as STAB1 in microglia and MAL in oligodendrocytes for Parkinsons disease (PD). Moreover, we dissect the complex inverted haplotype of the MAPT (encoding tau) PD risk locus, identifying ectopic enhancer-gene contacts in neurons that increase MAPT expression and may mediate this disease association. This work greatly expands our understanding of inherited variation in AD and PD and provides a roadmap for the epigenomic dissection of noncoding regulatory variation in disease.

genomics↗

bioRxiv2019

The GTEx Consortium atlas of genetic regulatory effects across human tissues

The Genotype-Tissue Expression (GTEx) project was established to characterize genetic effects on the transcriptome across human tissues, and to link these regulatory mechanisms to trait and disease associations. Here, we present analyses of the v8 data, based on 17,382 RNA-sequencing samples from 54 tissues of 948 post-mortem donors. We comprehensively characterize genetic associations for gene expression and splicing in cis and trans, showing that regulatory associations are found for almost all genes, and describe the underlying molecular mechanisms and their contribution to allelic heterogeneity and pleiotropy of complex traits. Leveraging the large diversity of tissues, we provide insights into the tissue-specificity of genetic effects, and show that cell type composition is a key factor in understanding gene regulatory mechanisms in human tissues.

genetics↗

bioRxiv2019

Systematic assessment of regulatory effects of human disease variants in pluripotent cells

Identifying regulatory genetic effects in pluripotent cells provides important insights into disease variants with potentially transient or developmental origins. Combining existing and newly-generated data, we characterized 1,367 iPSC lines from 948 unique donors, collectively analyzed within the \"Integrated iPSC QTL\" (i2QTL) Consortium. The sample size of our study allowed us to derive the most comprehensive map of quantitative trait loci (QTL) in pluripotent human cells to date. We mapped the effects of nearby common genetic variants on five expression phenotypes, identifying cis-QTL at gene-, exon-level and transcript-, splicing-, alternative polyadenylation-ratio (APA) for a total of 18,556 genes. For gene-level, we further quantified the effects of rare and singleton variants, and the effect of distal variants that act in trans (trans-eQTL), which we replicated in independent samples. Our data are a valuable community resource, uncovering novel regulatory effects that have not previously been described in differentiated cells and tissues. Building on this regulatory map, we functionally explore GWAS signals for over 4,336 trait loci, finding evidence for colocalization with common and rare iPSC QTL for traits such as height and BMI, and diseases, such as cancer and coronary artery disease.

genomics↗

bioRxiv2018

Identification of rare-disease genes in diverse undiagnosed cases using whole blood transcriptome sequencing and large control cohorts

RNA sequencing (RNA-seq) is a complementary approach for Mendelian disease diagnosis for patients in whom exome-sequencing is not informative. For both rare neuromuscular and mitochondrial disorders, its application has improved diagnostic rates. However, the generalizability of this approach to diverse Mendelian diseases has yet to be evaluated. We sequenced whole blood RNA from 56 cases with undiagnosed rare diseases spanning 11 diverse disease categories to evaluate the general application of RNA-seq to Mendelian disease diagnosis. We developed a robust approach to compare rare disease cases to existing large sets of RNA-seq controls (N=1,594 external and N=31 family-based controls) and demonstrated the substantial impacts of gene and variant filtering strategies on disease gene identification when combined with RNA-seq. Across our cohort, we observed that RNA-seq yields a 8.5% diagnostic rate. These diagnoses included diseases where blood would not intuitively reflect evidence of disease. We identified RARS2 as an under-expression outlier containing compound heterozygous pathogenic variants for an individual exhibiting profound global developmental delay, seizures, microcephaly, hypotonia, and progressive scoliosis. We also identified a new splicing junction in KCTD7 for an individual with global developmental delay, loss of milestones, tremors and seizures. Our study provides a broad evaluation of blood RNA-seq for the diagnosis of rare disease.

genomics↗