Biology subjects

Breuss, M. W.

Publications and source records attributed to Breuss, M. W..

9 recordsLinked to original sources

bioRxiv2025

Long-read sequencing reveals extensive FMR1 somatic mosaicism in Fragile-X associated tremor/ataxia syndrome in human brain

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a progressive neurodegenerative disorder caused by a CGG repeat expansion in the 5 untranslated region of the X-linked Fragile X messenger ribonucleoprotein 1 (FMR1). Although the CGG repeat tract is known for instability that has been posited to contribute to clinical heterogeneity, the extent of somatic variation in human brain remains unclear, in part due to the technical limitations of sequencing long tandem repeats. Here, we quantified FMR1 somatic variation in post-mortem brain tissue from individuals with FXTAS (n = 6) and Fragile X syndrome (FXS, n = 2) by applying amplification-free, targeted, long-read sequencing. This approach uncovered remarkable somatic mosaicism in repeat size and methylation in FXTAS, including somatic expansions and contractions which were not resolvable with traditional approaches. For example, in FXTAS, we identified somatic expansions to over 6000 base pairs in length as well as contractions to below the pathogenic range. We also identified unexpected patterns of methylation mosaicism on pre and full mutations. On the other hand, we replicated prior findings suggesting differential CGG expansion on the active X-chromosome in XX females. Finally, we examined the above cohort for expansions in 19 additional disease-associated repeat loci. Remarkably, we identified additional expansions in 5 out of 8 affected individuals, in FXN and RFC1. This work provides new insight into the extensive molecular variation underlying FXTAS in human brain and establishes a framework for studying repeat expansion disorders more broadly, highlighting the potential of long-read sequencing to advance our fundamental understanding of somatic mosaicism of these intractable regions of our genome.

genetics↗

bioRxiv2025

Exon-skipping and genetic compensation due to biallelic mutations in the neurodevelopmental disease gene LNPK

Homozygous loss-of-function mutations in LNPK, the gene encoding the endoplasmic reticulum-associated protein lunapark, have previously been linked to an autosomal recessive neurodevelopmental syndrome. Here, we describe an individual harboring compound heterozygous predicted splice site mutations with an overall matching phenotype. In cultured fibroblasts, these mutations result in a dearth of transcript and severe loss of protein, thereby establishing their likely pathogenicity. The underlying reduction in gene expression is due to the activation of the nonsense-mediated decay (NMD) pathway as a consequence of exon-skipping rather than intron retention, leading to aberrant transcripts. We further demonstrate that LNPK is subject to genetic compensation, as both cells from the affected individual and her mother exhibit a significant increase in transcript compared to a control cell line when treated with an inhibitor of NMD. Together, this report describes novel disease-causing variants in LNPK and reveals their impact on transcription and mRNA stability.

genetics↗

bioRxiv2025

CREPE (CREate Primers and Evaluate): a computational tool for large-scale primer design and specificity analysis

Polymerase chain reaction (PCR) is ubiquitous in biological research labs, as it is a fast, flexible, and cost-effective technique to amplify a DNA region of interest. However, manual primer design can be an error-prone and time-consuming process depending on the number and composition of target sites. While Primer3 has emerged as an accessible tool to solve some of these issues, additional computational pipelines are required for appropriate scaling. Moreover, this does not replace the manual confirmation of primer specificity (i.e., the assessment of off-targets). To overcome the challenges of large-scale primer design, we fused the functionality of Primer3 and In-Silico PCR (ISPCR); this integrated pipeline, CREPE (CREate Primers and Evaluate), performs primer design and specificity analysis through a custom evaluation script for any given number of target sites at scale. Its final output summarizes the lead forward and reverse primer pair for each target site, a measure of the likelihood of binding to off-targets, and additional information to aid decision-making. We provide this through a customized workflow for targeted amplicon sequencing (TAS) on a 150bp paired-end Illumina platform. Experimental testing showed successful amplification for more than 90% of primers deemed acceptable by CREPE.

genetics↗

bioRxiv2023

Cell-type-resolved somatic mosaicism reveals clonal dynamics of the human forebrain

Debate remains around anatomic origins of specific brain cell subtypes and lineage relationships within the human forebrain. Thus, direct observation in the mature human brain is critical for a complete understanding of the structural organization and cellular origins. Here, we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific Mosaic Variant Barcode Analysis. From four hemispheres from two different human neurotypical donors, we identified 287 and 780 mosaic variants (MVs), respectively that were used to deconvolve clonal dynamics. Clonal spread and allelic fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted compared with resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome-transcriptome analysis at both a cell-type-specific and single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of MVs across 17 locations within one parietal lobe reveals restrictions of clonal spread in the anterior-posterior axis precedes that of the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus cell-type resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

neuroscience↗

bioRxiv2022

Comprehensive multiomic profiling of somatic mutations in malformations of cortical development

Malformations of cortical development (MCD) are neurological conditions displaying focal disruption of cortical architecture and cellular organization arising during embryogenesis, largely from somatic mosaic mutations. Identifying the genetic causes of MCD has been a challenge, as mutations remain at low allelic fractions in brain tissue resected to treat epilepsy. Here, we report a genetic atlas from 317 brain resections, identifying 69 mutated genes through intensive profiling of somatic mutations, combining whole-exome and targeted-amplicon sequencing with functional validation and single-cell sequencing. Genotype-phenotype correlation analysis elucidated specific MCD gene sets associating distinct pathophysiological and clinical phenotypes. The unique spatiotemporal expression patterns identified by comparing single-nucleus transcriptional sequences of mutated genes in control and patient brains implicate critical roles in excitatory neurogenic pools during brain development, and in promoting neuronal hyperexcitability after birth.

genetics↗

bioRxiv2022

Sperm mosaicism predicts transmission of de novo mutations to human blastocysts

De novo mutations underlie individually rare but collectively common pediatric congenital disorders. Some of these mutations can also be detected in tissues and from cells in a parent, where their abundance and tissue distribution can be measured. We previously reported that a subset of these mutations is detectable in sperm from the father, predicted to impact the health of offspring. Here, in three independent couples undergoing in vitro fertilization, we first assessed male gonadal mosaicism, then assessed the transmission of the mutations to their preimplantation blastocysts. We found an overall predictable transmission but slight under-transmission of mutations to blastocysts based upon measured mutational abundance in sperm, and we replicated this conclusion in an independent family-based cohort. Therefore, unbiased preimplantation genetic testing for gonadal mosaicism may represent a feasible approach to reduce the transmission of potentially harmful de novo mutations, which could help to reduce their impact on miscarriage and pediatric disease.

genetics↗

bioRxiv2020

DeepMosaic: Control-independent mosaic single nucleotide variant detection using deep convolutional neural networks

Introductory paragraphMosaic variants (MVs) reflect mutagenic processes during embryonic development1 and environmental exposure2, accumulate with aging, and underlie diseases such as cancer and autism3. The detection of MVs has been computationally challenging due to sparse representation in non-clonally expanded tissues. While heuristic filters and tools trained on clonally expanded MVs with high allelic fractions are proposed, they show relatively lower sensitivity and more false discoveries4-9. Here we present DeepMosaic, combining an image-based visualization module for single nucleotide MVs, and a convolutional neural networks-based classification module for control-independent MV detection. DeepMosaic achieved higher accuracy compared with existing methods on biological and simulated sequencing data, with a 96.34% (158/164) experimental validation rate. Of 932 mosaic variants detected by DeepMosaic in 16 whole genome sequenced samples, 21.89-58.58% (204/932-546/932) MVs were overlooked by other methods. Thus, DeepMosaic represents a highly accurate MV classifier that can be implemented as an alternative or complement to existing methods.

genomics↗

bioRxiv2020

Temporal stability of human sperm mosaic mutations results in life-long threat of transmission to offspring

Every newborn harbors scores of new single nucleotide variants (SNVs) that may impact health and disease1-4; the majority of these are contributed by the paternal germ cells5. In some cases, these mutations are identifiable in a subset of the parents cells--a phenomenon called mosaicism, which is capable of producing disease recurrence6-8. Here, we provide a comprehensive analysis of male gonadal mosaic mutations, employing 300x whole genome sequencing (WGS) of blood and sperm in 17 healthy individuals, including assessment across multiple samples and age groups. Approximately 1 in 15 healthy males is predicted to harbor a transmissible, likely pathogenic exonic variant that is mosaic in his sperm. In general, only a third of sperm mosaic mutations were detectable in blood cells, all were remarkably stable over the course of months to years, and 23% were present in 5% or more of sperm cells. There was no evidence of age-dependent clonal expansion or collapse, as seen in hematopoiesis. Thus, despite the observed increase of mutations in offspring of men with advanced paternal age, detectable sperm mosaicism remains stable, represents a life-long transmission risk to offspring, and suggests a testis stem cell niche that prevents widespread clonality.

genomics↗

bioRxiv2020

Somatic mosaicism in the mature brain reveals clonal cellular distributions during cortical development

The structure of the human neocortex underlies species-specific features and is a reflection of intricate developmental programs. Here we analyzed neocortical cellular lineages through a comprehensive assessment of brain somatic mosaicism--which acts as a neutral recorder of lineage history. We employed deep whole genome and variant sequencing in a single postmortem neurotypical human brain across 25 anatomic regions and three distinct modalities: bulk geographies, sorted cell types, and single nuclei. We identified 259 mosaic variants, revealing remarkable differences in localization, clonal abundance, cell type specificity, and clade distribution. We identified a set of hierarchical cellular diffusion barriers, whereby the left-right axis separation of the neocortex occurs prior to anterior-posterior and dorsal-ventral axis separation. We also found that stochastic distribution is a driver of clonal dispersion, and that rules regarding cellular lineages and anatomical boundaries are often ignored. Our data provides a comprehensive analysis of brain somatic mosaicism across the human cerebral cortex, deconvolving clonal distributions and migration patterns in the human embryo. One Sentence SummaryComprehensive evaluation of brain somatic mosaicism in the adult human identifies rules governing cellular distribution during embryogenesis.

genomics↗