Biology subjects

Richman, J.

Publications and source records attributed to Richman, J..

9 recordsLinked to original sources

bioRxiv2026

Large-scale Perturbation of Systems Biology-Derived Genes Reveals Modifiers of HD-associated Transcriptomic Networks and Pathology

In Huntingtons disease (HD), disease-driver genes are broadly expressed, but pathogenic players specific to vulnerable neurons remain poorly defined. Leveraging a previously defined mutant huntingtin (mHtt) CAG-length-associated gene network, we perturbed 115 module hub genes with heterozygous knockout (KO-het) to assess their striatal transcriptomic modifier effects in wildtype and Q140 HD mice. We generated 3,592 striatal RNA-seq datasets, mapped 6,517 perturbagen-responder gene pairs, and uncovered regulators of medium spiny neuron (MSN) identity gene expression and DNA- methylase/demethylase-sensitive genes. We developed a bioinformatic pipeline to rank the perturbations with significant impacts on striatal transcriptome and HD-associated gene networks in wildtype or Q140 mice. KO-het for FoxP1 and Scn4b (two MSN-selective genes) exacerbated, whereas Pdp1 KO-het ameliorated, Q140 striatal pathology. Importantly, knockdown of functionally opposing ion channels, SCN4B and KCNH4, dichotomously affected aggregation and neurodegeneration in reprogrammed HD patient MSNs. Together, our study rigorously evaluated systems biology-derived candidates to identify modifiers of HD-associated molecular networks and pathology, providing an in vivo perturbation- transcriptome resource and highlighting genes involved in MSN excitability, transcription, calcium signaling, and mitochondrial metabolism in HD pathogenesis.

genetics↗

bioRxiv2026

The abnormal C-terminus in DVL1 impacts Robinow Syndrome phenotypes

Robinow Syndrome is a polygenic, rare skeletal disorder characterized by craniofacial and limb defects. The genes involved are in the Wingless-related Integration site-1 (WNT) pathway and DVL1 (Dishevelled 1) is the most commonly affected gene. In all pathogenic variants of DVL1, a frameshift replaces the C terminus with a novel peptide. We tested whether the variant DVL11519{Delta}T was sufficient to alter development in vivo and in vitro in two animal models. We compared phenotypes to wtDVL1 or DVL1 with a stop codon at position 1519. Misexpression of DVL11519{Delta}T in the developing face of chicken embryos with an avian retrovirus, leads to a widening of the frontonasal mass similar to the human facial phenotype and ultimately to inhibition of skeletogenesis that was also verified in primary cultures of frontonasal mass mesenchyme. In luciferase assays carried out in facial mesenchyme, the wtDVL1 activated canonical and JNK PCP WNT signalling however the DVL11519* and the DVL11519{Delta}T variant removed some but not all of the signaling activity. We also determined that there is mislocalization of the protein expressed from DVL11519{Delta}T in the nucleus while the other two constructs were mainly found in the cytoplasm. In complementary Drosophila experiments using a variety of readouts, only the DVL11519{Delta}T variant impacted morphogenesis and signaling. This is the first study to clarify the pathogenesis of Robinow syndrome is due to the novel C-terminus of DVL1 which exerts dominant interference on morphogenesis, skeletogenesis and WNT signaling.

developmental biology↗

bioRxiv2024

Robinow Syndrome DVL1 mutations disrupt morphogenesis and appendage formation in a Drosophila disease model

Robinow Syndrome is a rare developmental syndrome caused by variants in numerous genes involved in Wnt signaling pathways. We previously showed that expression of patient variants in Drosophila and a chicken model disrupts the balance of canonical and non-canonical/PCP Wnt signaling. We also noted neomorphic effects that warranted further investigation. In this study, we examine morphological changes that occur as a result of one variant, DVL11519{Delta}T, that serves as a prototype for the other pathogenic variants. We show that epithelial imaginal disc development is disrupted in legs and wings. Shortened leg segments are reminiscent of shortened limb bones seen in RS patients. We find that imaginal disc development is disrupted and accompanied by increased cell death, without changes in cell proliferation. Furthermore, we find altered dynamics of basement membrane components and modulators. Notably we find increased MMP1 expression and tissue distortion, which is dependent on JNK signaling. We also find enhanced collagen IV (Viking) secreted from cells expressing DVL11519{Delta}T. Through these studies we have gained more insight into developmental consequences of DVL1 variants implicated in autosomal dominant Robinow Syndrome.

developmental biology↗

bioRxiv2024

DVL1 variants and C-terminal deletions have differential effects on craniofacial development and WNT signaling

Robinow Syndrome (RS) is a rare disease characterized by craniofacial malformations and limb shortening linked with mutations in seven WNT pathway genes. Our objective was to investigate the functional effects of frameshift mutations the intracellular adaptor protein, Dishevelled (DVL1; c.1519{Delta}T, p.Trp507Glyfs*142) on chicken craniofacial development. Misexpression of wt (wt) or mutant hDVL1 variants in vivo caused upper beak shortening (wtDVL1 n=8/14; DVL11519{Delta}T 12/13). At early stages of development, the DVL11519{Delta}T inhibited frontonasal mass narrowing, chondrogenesis, and proliferation. To test whether the phenotypes were caused due to the abnormal C-terminal peptide in DVL11519{Delta}T, we designed two additional constructs. The DVL11519* (DVL1507*) retains first 30 amino acids of the C-terminus while DVL11431* (DVL1477*) removes the entire C-terminus. DVL11519* injected embryos had normal beaks while DVL11431* caused high mortality and the phenotypes were like the DVL11519{Delta}T. In frontonasal micromass cultures, both DVL11519{Delta}T and DVL11431* inhibited skeletogenesis while the DVL11519* resembled wtDVL1 and GFP cultures. In luciferase assays DVL11519{Delta}T, DVL11519*and DVL11431* weakly activated the WNT canonical and non-canonical JNK-PCP pathways compared to wtDVL1. Furthermore, we observed that variant DVL1507*fs is stalled in the nucleus similar to hDVL1477*, possibly due to the abnormal C-terminus interfering with the nuclear export sequence. wtDVL1 and DVL1507* were distributed in nucleus and the cytoplasm. Our RS-DVL11519{Delta}T avian model recapitulates the broad face and jaw hypoplasia and demonstrates defects in both branches of WNT signaling. This is the first study to clarify the role of abnormal C-terminus in ADRS and to recognize the importance of an uncharacterized C-terminal sequence. Summary StatementFunctional and biochemical studies on chicken embryos with the Robinow syndrome (RS) DVL1 variant demonstrate defects in skeletogenesis and both branches of WNT signaling. This is the first study to establish a link between the RS facial defects and the mutated C-terminal sequence. We identified first 30 amino acids of the DVL1 C-terminus are sufficient for normal development.

developmental biology↗

bioRxiv2023

Functional genomics in chicken embryos reveal the pathogenicity of two missense FZD2 variants associated with dominant Robinow syndrome

Robinow syndrome (RS) is a rare disease caused by mutations in seven WNT pathway genes. Features include craniofacial widening and jaw hypoplasia. We used the chicken embryo to test two autosomal dominant RS (ADRS) missense FZD2 variants on the frontonasal mass, the affected region in RS. The wild-type (wt) and variant hFZD2 inhibited beak ossification. The bone hypoplasia was possibly mediated by decreased levels of WNT and BMP pathway genes. In primary cultures, hFZD2 variants inhibited chondrogenesis, increased nuclear shuttling of {beta}-catenin and increased expression of TWIST1, both known to suppress chondrogenesis. In luciferase reporter assays, proteins coding for 1301G>T and 425C>T FZD2 variants weakly activated canonical WNT reporter and dominantly interfered with wtFZD2. In the JNK-PCP WNT pathway luciferase assay, only the 425C>T showed a loss-of-function. The 1301G>T variant presumably acts through a JNK-independent pathway. This is the first study to demonstrate that the ADRS-FZD2 missense variants cause craniofacial and WNT signaling defects. Frontonasal mass width is increased by both hFZD2 variants which sheds light on the ontogeny of the broad facial features seen in individuals with RS. Summary StatementGain-of-function studies on FZD2 missense variants associated with Robinow syndrome led to increased facial width, altered Wnt signaling and inhibition of beak skeletogenesis in chicken embryos.

developmental biology↗

bioRxiv2023

BAC Transgenic Expression of Human TREM2-R47H Remodels Amyloid Plaques but Unable to Reprogram Plaque-associated Microglial Reactivity in 5xFAD Mice

BackgroundGenetic study of late-onset Alzheimers disease (AD) reveals that a rare Arginine-to-Histamine mutation at amino acid residue 47 (R47H) in Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) results in increased disease risk. TREM2 plays critical roles in regulating microglial response to amyloid plaques in AD, leading to their clustering and activation surrounding the plaques. We previously showed that increasing human TREM2 gene dosage exerts neuroprotective effects against AD-related deficits in amyloid depositing mouse models of AD. However, the in vivo effects of the R47H mutation on human TREM2-mediated microglial reprogramming and neuroprotection remains poorly understood. MethodHere we created a BAC transgenic mouse model expressing human TREM2 with the R47H mutation in its cognate genomic context (BAC-TREM2-R47H). Importantly, the BAC used in this study was engineered to delete critical exons of other TREM-like genes on the BAC to prevent confounding effects of overexpressing multiple TREM-like genes. We crossed BAC-TREM2- R47H mice with 5xFAD [1], an amyloid depositing mouse model of AD, to evaluate amyloid pathologies and microglial phenotypes, transcriptomics and in situ expression of key TREM2-dosage dependent genes. We also compared the key findings in 5xFAD/BAC-TREM2-R47H to those observed in 5xFAD/BAC-TREM2 mice. ResultBoth BAC-TREM2 and BAC-TREM2-R47H showed proper expression of three splicing isoforms of TREM2 that are normally found in human. In 5xFAD background, elevated TREM2-R47H gene dosages significantly reduced the plaque burden, especially the filamentous type. The results were consistent with enhanced phagocytosis and altered NLRP3 inflammasome activation in BAC- TREM2-R47H microglia in vitro. However, unlike TREM2 overexpression, elevated TREM2- R47H in 5xFAD failed to ameliorate cognitive and transcriptomic deficits. In situ analysis of key TREM2-dosage dependent genes and microglial morphology uncovered that TREM2-R47H showed a loss-of-function phenotype in reprogramming of plaque-associated microglial reactivity and gene expression in 5xFAD. ConclusionOur study demonstrated that the AD-risk variant has a previously unknown, mixture of partial and full loss of TREM2 functions in modulating microglial response in AD mouse brains. Together, our new BAC-TREM2-R47H model and prior BAC-TREM2 mice are invaluable resource to facilitate the therapeutic discovery that target human TREM2 and its R47H variant to ameliorate AD and other neurodegenerative disorders.

neuroscience↗

bioRxiv2018

Measures of reproducibility in sampling and laboratory processing methods in high-throughput microbiome analysis

Microbial community analysis can be biased by multiple technical factors, such as storage conditions, DNA extraction, or amplification conditions. In a high-throughput laboratory that relies on samples obtained from thousands of different subjects, knowledge of the extent of subject-introduced sampling and storage variation on the outcome of the inferred microbiome, as well as the effect of laboratory-introduced variation caused by reagent batches, equipment, or operator on the consistency of these processes within the laboratory is paramount. Here, we analyzed the effect of sampling from different parts of the same stool specimen or on different consecutive days, as well as short-term storage of samples at different temperatures on microbiome profiles obtained by 16S rRNA gene amplification. Each of these factors had relatively little effect on the microbial composition. In addition, replicate amplification of 44 stool samples showed reproducible results. Finally, 363 independent replicate extractions and amplifications of a single human homogenized stool (HS) specimen showed reproducible results (average Lins correlation = 0.95), with little variation introduced by HS batch, operator, extraction equipment, or DNA sequencer. In all cases, variations between replicates were significantly smaller than those between individual samples; subject identity always was the largest determinant. We propose that homogenized stool specimens could be used as quality control to routinely monitor the laboratory process and to validate new methods.

microbiology↗

bioRxiv2017

A new sequencing-based women’s health assay combining self-sampling, HPV detection and genotyping, STI detection, and vaginal microbiome analysis

The composition of the vaginal microbiome, including both the presence of pathogens involved in sexually transmitted infections (STI) as well as commensal microbiota, has been shown to have important associations for a womans reproductive and general health. Currently, healthcare providers cannot offer comprehensive vaginal microbiome screening, but are limited to the detection of individual pathogens, such as high-risk human papillomavirus (hrHPV), the predominant cause of cervical cancer. There is no single test on the market that combines HPV, STI, and microbiome screening. Here, we describe a novel inclusive womens health assay that combines self-sampling with sequencing-based HPV detection and genotyping, vaginal microbiome analysis, and STI-associated pathogen detection. The assay includes genotyping and detection of 14 hrHPV types, 5 low-risk HPV types (lrHPV), as well as the relative abundance of 32 bacterial taxa of clinical importance, including Lactobacillus, Sneathia, Gardnerella, and 4 pathogens involved in STI, with high sensitivity, specificity, and reproducibility. For each of these taxa, healthy ranges were determined in a group of 50 self-reported healthy women. The hrHPV portion of the test was evaluated against the Digene High-Risk HPV HC2 DNA test with vaginal samples obtained from 185 women. Results were concordant for 181/185 of the samples (overall agreement of 97.83%, Cohens kappa = 0.93), with sensitivity and specificity values of 94.74% and 98.64%, respectively. Two discrepancies were caused by the Digene assays known cross-reactivity with low-risk HPV types, while two additional samples were found to contain hrHPV not detected by Digene. This novel assay could be used to complement conventional cervical cancer screening, because its self-sampling format can expand access among women who would otherwise not participate, and because of its additional information about the composition of the vaginal microbiome and the presence of pathogens.

microbiology↗

bioRxiv2016

16S rRNA Gene Sequencing as a Clinical Diagnostic Aid for Gastrointestinal-related Conditions

Accurate detection of the microorganisms underlying gut dysbiosis in the patient is critical to initiate the appropriate treatment. However, most clinical microbiology techniques used to detect gut bacteria were developed over a century ago and rely on culture-based approaches that are often laborious, unreliable, and subjective. Further, culturing does not scale well for multiple targets and detects only a minority of the microorganisms in the human gastrointestinal tract. Here we present a clinical test for gut microorganisms based on targeted sequencing of the prokaryotic 16S rRNA gene. We tested 46 clinical prokaryotic targets in the human gut, 28 of which can be identified by a bioinformatics pipeline that includes sequence analysis and taxonomic annotation. Using microbiome samples from a cohort of 897 healthy individuals, we established a reference range defining clinically relevant relative levels for each of the 28 targets. Our assay accurately quantified all 28 targets and correctly reflected 38/38 verification samples of real and synthetic stool material containing known pathogens. Thus, we have established a new test to interrogate microbiome composition and diversity, which will improve patient diagnosis, treatment and monitoring. More broadly, our test will facilitate epidemiological studies of the microbiome as it relates to overall human health and disease.

microbiology↗