Biology subjects

Shaw, G.

Publications and source records attributed to Shaw, G..

14 recordsLinked to original sources

bioRxiv2026

Spatial transcriptomics defines the mechanisms of hiPSC derived stem cell mediated repair in human articular cartilage

We here determined therapeutic efficacy and mode-of-action of human induced pluripotent-derived therapeutic stem cells (hiMSCs) across in vivo mouse and ex vivo human osteoarthritis models. hiMSC treatment in DMM-mice significantly reduced OARSI damage scores, which was affirmed by a decrease in the catabolic marker Mmp13 and an increase in the anabolic marker Col2. These treatment effects appeared, irrespective of modifying factors such as xeno-free media or thermosensitive hydrogel carrier. Subsequently treatment of hiMSC+gel in human osteoarthritic cartilage explants showed a transcriptome-wide significant activation of the cholesterol and sterol synthesis pathways marked by genes such as MVD, DHCR7, MSMO1, FABP3. Additionally, we showed that these changes alleviated OA-associated imbalances of the cellular Zinc-ion homeostasis pathways, represented by genes such as MT1F, MT1G, MT1H and SLC30A1. Spatial transcriptomics then sensitively captured that hiMSC+gel treatment evoked, specifically at the superficial cartilage layer, a consistent upregulation of healthy chondrocyte markers such as CHAD, ACAN, FRZB, and SOX9, alongside a suppression of catabolic and inflammatory mediators such as SERPINE1, SPP1, MMP13, ADAMTS5. Our findings link therapeutic outcomes of hiMSC treatment to precise spatially resolved molecular changes in human tissue, that would otherwise be obscured by heterogeneous cell populations. Collectively our study highlighted that hiPSC-derived stem cell therapy (hiMSCs) could provide a scalable off-the-shelf solution to treat osteoarthritis, with strong prospects for clinical applications in the near future.

developmental biology↗

bioRxiv2025

Getting a head start: Craniofacial heterochrony in marsupials involves dynamic changes to molecular and cellular mechanisms underlying neural crest development

The neural crest is a vertebrate innovation central to craniofacial development and evolution. While the gene regulatory networks guiding neural crest development are well characterized, the mechanisms generating species-specific craniofacial diversity remain poorly understood. Marsupials provide a unique model for studying neural crest plasticity, having evolved accelerated patterns of craniofacial development during embryogenesis. This adaptation arises in response to marsupials being born altricial after a short gestation yet require well-developed mouthparts to attach to a teat and continue development in the pouch. However, how marsupials achieve this heterochronic shift in neural crest development is largely unknown. In this study, we investigate the cellular and molecular mechanisms underlying their distinct heterochrony, revealing that marsupials produce dense pre-migratory aggregates of neural crest cells which undergo collective migration as epithelial-like sheets, potentially facilitating rapid establishment of the facial prominences. These cellular behaviours are unique amongst amniotes but resemble patterns in anamniotes which similarly exhibit accelerated craniofacial development to support early feeding. Marsupials appear to have evolved a similar mechanism of neural crest migration to facilitate their developmental heterochrony. These findings suggest that vertebrate neural crest migration may be shaped by the pace of craniofacial development during embryogenesis rather than phylogeny, providing new perspectives on neural crest plasticity and the developmental mechanisms driving craniofacial diversity across vertebrates.

developmental biology↗

bioRxiv2025

Therapeutic efficacy of hiMSC-derived Extracellular Vesicles from Serum-containing and Xeno-free media for osteoarthritis treatment

BackgroundExtracellular vesicles derived from human induced mesenchymal stromal cells (hiEVs) constitute a promising cell-free therapeutic option for osteoarthritis. To allow translation to the clinic we evaluated the therapeutic effects of hiEVs for osteoarthritis treatment. Specifically, we assessed the efficacy of hiEVs collected from serum-containing and serum-free, PurStem (PS), media in an OA mice model. MethodshiEVs were administered with or without hydrogel via intra-articular (i.a.) injection in a destabilization of the medial meniscus (DMM) mouse model. Fluorescence imaging was used to monitor the retention of IR780-labeled hiEVs in the joint cavity. The therapeutic effects were evaluated by analyzing damage scores as well as catabolic and anabolic markers, including Mmp13 and Col2 expression, in joint tissues. ResultsFluorescence imaging confirmed that hiEVs remained localized at the injection site without systemic migration. HiEVs demonstrated significant protective effects against joint tissue degeneration in the DMM mouse OA model, as evidenced by reduced damage scores, decreased Mmp13 expression, and increased anabolic processes (Col2 expression). The hydrogel alone also exerted beneficial therapeutic effects, including reduced damage scores, increased Col2 expression, and reduced Mmp13 levels; however, these effects were notably smaller than those achieved with hiEV treatment while it was independent of the medium used for hiEV collection. ConclusionsTogether, our findings demonstrate that hiEVs from xeno-free conditions effectively prevent cartilage degradation and promote its repair. This paves the way for future clinical translation of hiEV-based therapies as a safe, scalable, and effective approach to treat osteoarthritis.

developmental biology↗

bioRxiv2025

GFAP Proteolysis in TBI: Linking Novel Modified Cleavage Products to Astrocyte Pathology and Patient Outcomes

Glial fibrillary acidic protein (GFAP) is a significant clinical biomarker of traumatic brain injury (TBI), yet understanding the nature, timing, and impact of its degraded and modified products would inform clinical utility. We report novel GFAP breakdown products (BDPs) and post-translational modifications (PTMs) that are unique to TBI including fragment- and patient-specific citrullination signatures that destabilize GFAP filaments. GFAP and its fragments were sequenced by mass spectrometry (MS) from severe TBI patients cerebrospinal fluid (CSF) and sera, identifying two distinct TBI-specific jointly generated product sets within the rod-domain covering coil1 (20-26kDa) and coil2 (15-19kDa). Their endings differed from GFAP fragments described in Alzheimers and Alexander disease. Label-free quantification showed biofluid co-product abundance differences with coil1-BDPs enriched over coil2-BDPs. Coil imbalance was independently confirmed by immunoblot densitometry in twenty-three TBI patients. Measurements over ten days showed injury day peaks of full-length and end-clipped GFAP fragments, while proteolytic 37/39kDa and small fragments remained elevated. Six-month outcome (Extended Glasgow Outcome Scale) correlated with GFAP fragments, whereas levels of uncleaved and end-clipped GFAP did not. A human astrocyte culture trauma model provided mechanistic insights linking fluid GFAP levels, subcellular localization, and injury-induced astrocytopathy. A new cleavage site between the two coils was identified through selective epitope loss. Coil1-BDPs were fluid-released post-injury, while coil2-BDPs remained intracellular. Their cellular retention was explained by non-filamentous aggregation of citrulline-modified coil2-BDPs in pathological astrocytes. Trauma-triggered proteolysis involved calpains and caspases in specific astrocyte injury states using protease inhibitors and live-dye-reporter imaging. These novel data link TBI biofluid GFAP fragments with assembly-defective GFAP aggregation in pathological astrocytes. Clinical TBI outcome correlated with GFAP degradation rather than with overall GFAP release. These translational findings indicate that TBI biomarker GFAP undergoes degradation and modification alongside astrocyte pathology, providing a new conceptual framework for investigating biomarker-associated astrocyte proteinopathy potentially linked to post-traumatic neurodegeneration.

neuroscience↗

bioRxiv2024

Assessing the impact of autologous neutralizing antibodies on rebound dynamics in postnatally SHIV-infected ART-treated infant Rhesus macaques

AO_SCPLOWBSTRACTC_SCPLOWThe presence of antibodies against HIV in infected children is associated with a greater capacity to control viremia in the absence of therapy. While the benefits of early antiretroviral treatment (ART) in infants are well documented, early ART may interfere with the development of antibody responses. In contrast to adults, early treated children lack detectable HIV-specific antibodies, suggesting a fundamental difference in HIV pathogenesis. Despite this potential adverse effect, early ART may decrease the size of the latent reservoir established early in infection in infants, which can be beneficial in viral control. Understanding the virologic and immunologic aspects of pediatric HIV is crucial to inform innovative targeted strategies for treating children living with HIV. In this study, we investigate how ART initiation time sets the stage for trade-offs in the latent reservoir establishment and the development of humoral immunity and how these, in turn, affect post-treatment dynamics. We also elucidate the biological function of antibodies in pediatric HIV. We employ mathematical modeling coupled with experimental data from an infant nonhuman primate Simian/Human Immunodeficiency Virus (SHIV) infection model. Infant Rhesus macaques (RMs) were orally challenged with SHIV.C.CH505 375H dCT four weeks after birth and started treatment at different times after infection. In addition to viral load measurements, antibody responses and latent reservoir sizes were measured. We estimate model parameters by fitting viral load measurements to the standard HIV viral dynamics model within a nonlinear fixed effects framework. This approach allows us to capture differences between rhesus macaques (RMs) that develop antibody responses or exhibit high latent reservoir sizes compared to those that do not. We find that neutralizing antibody responses are associated with increased viral clearance and decreased viral infectivity but decreased death rate of infected cells. In addition, the presence of detectable latent reservoir is associated with less robust immune responses. These results demonstrate that both immune response and latent reservoir dynamics are needed to understand post-rebound dynamics and point to the necessity of a comprehensive approach in tailoring personalized medical interventions.

immunology↗

bioRxiv2023

Divergent patterns of meiotic double strand breaks and synapsis initiation dynamics suggest an evolutionary shift in the meiosis program between American and Australian marsupials

In eutherian mammals, hundreds of programmed DNA double-strand breaks (DSBs) are generated at the onset of meiosis. The DNA damage response is then triggered. Although the dynamics of this response is well studied in eutherian mammals, recent findings have revealed different patterns of DNA damage signaling and repair in marsupial mammals. To better characterize these differences, here we analyzed synapsis and the chromosomal distribution of meiotic DSBs markers in three different marsupial species (Thylamys elegans, Dromiciops gliorides, and Macropus eugenii) that represent South American and Australian Orders. Our results revealed inter-specific differences in the chromosomal distribution of DNA damage and repair proteins, which were associated with differing synapsis patterns. In the American species T. elegans and D. gliroides, synapsis progressed exclusively from the chromosomal ends towards interstitial regions. This was accompanied by sparse H2AX phosphorylation, mainly accumulating at chromosomal ends, which appeared conspicuously polarized in a bouquet configuration at early stages of prophase I. Accordingly, RAD51 and RPA were mainly localized at chromosomal ends throughout prophaseI in both American marsupials, likely resulting in reduced recombination rates at interstitial positions. In sharp contrast, synapsis initiated at both interstitial and distal chromosomal regions in the Australian representative M. eugenii, {gamma}H2AX had a broad nuclear distribution, and RAD51 and RPA foci displayed an even chromosomal distribution. Given the basal evolutionary position of T. elegans, it is likely that the meiotic features reported in this species represent an ancestral pattern in marsupials and that a shift in the meiotic program occurred after the split of D. gliroides and the Australian marsupial clade. Our results open intriguing questions about the regulation and homeostasis of meiotic DSBs in marsupials. The low recombination rates observed at the interstitial chromosomal regions in American marsupials can result in the formation of large linkage groups, thus having an impact in the evolution of their genomes.

genetics↗

bioRxiv2022

Adaptation of a transmitted/founder simian-human immunodeficiency virus for enhanced replication in rhesus macaques

Transmitted/founder (TF) simian-human immunodeficiency viruses (SHIVs) express HIV-1 envelopes modified at position 375 to efficiently infect rhesus macaques while preserving authentic HIV-1 Env biology. TF SHIV.C.CH505 is an extensively characterized virus shown to recapitulate key features of HIV-1 immunobiology, including CCR5-tropism, a tier 2 neutralization profile, reproducible early viral kinetics, and authentic immune responses. SHIV.C.CH505 is used frequently in nonhuman primate studies of HIV, but viral loads after months of infection are variable and typically lower than those in people living with HIV. We hypothesized that additional mutations besides {Delta}375 might further enhance virus fitness without compromising essential components of CH505 Env biology. From sequence analysis of SHIV.C.CH505-infected macaques across multiple experiments, we identified a signature of envelope mutations associated with higher viremia. We then used short-term in vivo mutational selection and competition to identify a minimally adapted SHIV.C.CH505 with just five amino acid changes that substantially improve virus replication fitness in macaques. Next, we validated the performance of the adapted SHIV in vitro and in vivo and identified the mechanistic contributions of selected mutations. In vitro, the adapted SHIV shows improved virus entry, enhanced replication on primary rhesus cells, and preserved neutralization profiles. In vivo, the minimally adapted virus rapidly outcompetes the parental SHIV with an estimated growth advantage of 0.14 days-1 and persists through suppressive antiretroviral therapy to rebound at treatment interruption. Here, we report the successful generation of a well-characterized, minimally adapted virus, termed SHIV.C.CH505.v2, with enhanced replication fitness and preserved native Env properties that can serve as a new reagent for NHP studies of HIV-1 transmission, pathogenesis, and cure. Author SummaryThe power of the nonhuman primate model of HIV to predict outcomes in people living with HIV (PLWH) depends on authentic virus-host interactions. In pursuit of viruses that generate infection that mirrors the effects of HIV-1 in PLWH, we developed a minimally adapted version of a commonly used virus, SHIV.C.CH505, which has better fitness than the parental virus while retaining important biological properties. First, we studied virus sequences from SHIV.C.CH505-infected rhesus macaques to identify a signature of mutations common to animals with higher viral loads. We then tested viruses containing the various mutations in the lab and in animals to determine the most fit version and to identify the contribution of each mutation. Ultimately, we identified a minimally adapted version of SHIV.C.CH505 with just 5 amino acid substitutions that enhances virus replication and preserves CH505 envelope properties, including sensitivity to clinically relevant broadly neutralizing antibodies. This new virus, called SHIV.C.CH505.v2 replicates well in macaques over time and persists through antiretroviral therapy. SHIV.C.CH505.v2 could be an important component of nonhuman primate studies of HIV prevention, therapy, and cure.

microbiology↗

bioRxiv2022

Uman Type NF-L Antibodies Are Effective Reagents for the Imaging of Neurodegeneration

Recent work shows that certain immunological assays for the neurofilament light chain NF-L detect informative signals in the CSF and blood of human and animals affected by a variety of CNS injury and disease states. Much of this work has been performed using two mouse monoclonal antibodies to NF-L, UD1 and UD2, also known as 2.1 and 47.3 respectively. These are the essential components of the Uman Diagnostics NF-Light ELISA kit, the Quanterix Simoa bead based NF-L assay and others. We show here that the antibodies bind to neighboring epitopes in a short, conserved and unusual peptide in the NF-L "rod" Coil 2 region. We also describe a surprising and useful feature of Uman and similar reagents. While other well characterized NF-L antibodies show robust staining of countless cells and processes in CNS sections from healthy rats, both Uman antibodies reveal only a minor subset of presumably spontaneously degenerating or degenerated neurons and their processes. However following experimental mid-cervical injuries to rat spinal cord both Uman antibodies recognize numerous profiles in tissue sections. The Uman positive material was associated with fiber tracts expected to be damaged by the injury administered and the profiles had the swollen, beaded, discontinuous and sinusoidal morphology expected for degenerating and degenerated processes. We also found that several antibodies to the C terminal "tail" region of NF-L stain undamaged axonal profiles but fail to recognize the Uman positive material. The unmasking of the Uman epitopes and the loss of the NF-L tail epitopes can be mimicked by treating sections from healthy animals with proteases suggesting that the immunological changes we have discovered are due to neurodegeneration induced proteolysis. We have also generated a novel panel of monoclonal and polyclonal antibody reagents directed against the region of NF-L including the Uman epitopes which have staining properties identical to the Uman reagents. Using these we show that the NF-L region to which the Uman reagents bind contains further hidden epitopes distinct from those recognized by the two Uman reagents. We speculate that the Uman type epitopes are part of a binding region important for higher order neurofilament assembly. The work provides important insights into the properties of the NF-L biomarker, describes novel and useful properties of Uman type and NF-L tail binding antibodies and provides a hypothesis relevant to further understanding of neurofilament assembly.

neuroscience↗

bioRxiv2021

Cocaine-induced locomotor activation differs across six sets of inbred mouse substrains

Cocaine use disorders (CUD) are devastating for affected individuals and impose a significant burden on society, but there are currently no FDA-approved therapies. The development of novel and effective treatments has been hindered by substantial gaps in our knowledge about the etiology of these disorders. The risk for developing a CUD is influenced by genetics, the environment and complex interactions between the two. Identifying specific genes and environmental risk factors that increase CUD risk would provide an avenue for the development of novel treatments. Rodent models of addiction-relevant behaviors have been a valuable tool for studying the genetics of response to drugs of abuse. Traditional genetic mapping using genetically and phenotypically divergent inbred mice has been successful in identifying numerous chromosomal regions that influence addiction-relevant behaviors, but these strategies rarely result in identification of the causal gene or genetic variant. To overcome this challenge, reduced complexity crosses (RCC) between closely related inbred mouse substrains have been proposed as a method for rapidly identifying and validating functional variants. The RCC approach is dependent on identifying phenotypic differences between substrains. To date, however, the study of addiction-relevant behaviors has been limited to very few sets of substrains, mostly comprising the C57BL/6 lineage. The present study expands upon the current literature to assess cocaine-induced locomotor activation in 20 inbred mouse substrains representing six inbred strain lineages (A/J, BALB/c, FVB/N, C3H/He, DBA/2 and NOD) that were either bred in-house or supplied directly by a commercial vendor. To our knowledge, we are the first to identify significant differences in cocaine-induced locomotor response in several of these inbred substrains. The identification of substrain differences allows for the initiation of RCC populations to more rapidly identify specific genetic variants associated with acute cocaine response. The observation of behavioral profiles that differ between mice generated in-house and those that are vendor-supplied also presents an opportunity to investigate the influence of environmental factors on cocaine-induced locomotor activity.

genetics↗

bioRxiv2021

Transcriptional profiling of early differentiation of primary human mesenchymal stem cells into chondrocytes

Articular cartilage has only very limited regenerative capacities in humans. Tissue engineering techniques for cartilage damage repair are limited in the production of hyaline cartilage. Mesenchymal stem/stromal cells (MSCs) are multipotent stem cells and can be differentiated into mature cartilage cells, chondrocytes, which could be used for repairing damaged cartilage. Chondrogenesis is a highly complex, relatively inefficient process lasting over 3 weeks in vitro. In order to better understand chondrogenic differentiation, especially the commitment phase, we have performed transcriptional profiling of MSC differentiation into chondrocytes from early timepoints starting 15 minutes after induction to 16 hours and fully differentiated chondrocytes at 21 days. Transcriptional gene regulatory networks (GRN) were identified using time-course clustering and upstream-regulator predictions which revealed that cells start epigenetic reprogramming as early as 2 hours after induction and commit to differentiation within 4-6 hours. After that they adapt their gene expression to cater for differentiation specific protein production. These results suggest that interventions to improve the frequency and efficiency of differentiation should target early processes.

molecular biology↗

bioRxiv2021

TNF increases Tyrosine Hydroxylase expression in human monocytes

Most, if not all, peripheral immune cells in humans and animals express tyrosine hydroxylase (TH), the rate limiting enzyme in catecholamine synthesis. Since TH is typically studied in the context of brain catecholamine signaling, little is known about changes in TH production and function in peripheral immune cells. This knowledge gap is due, in part, to the lack of an adequately sensitive assay to measure TH in immune cells expressing lower TH levels compared to other TH expressing cells. Here, we report the development of a highly sensitive and reproducible Bio-ELISA to quantify picogram levels of TH in multiple model systems. We have applied this assay to monocytes isolated from blood of persons with Parkinsons disease (PD) and to age-matched, healthy controls. Our study unexpectedly revealed that PD patients monocytes express significantly higher levels of TH protein in peripheral monocytes relative to healthy controls. Tumor necrosis factor (TNF{delta}), a pro-inflammatory cytokine, has also been shown to be increased in the brains and peripheral circulation in human PD, as well as in animal models of PD. Therefore, we investigated a possible connection between higher levels of TH protein and the known increase in circulating TNF{delta} in PD. Monocytes isolated from healthy donors were treated with TNF{delta} or with TNF{delta} in the presence of an inhibitor. Tissue plasminogen activator (TPA) was used as a positive control. We observed that TNF{delta} stimulation increased both the number of TH+ monocytes and the quantity of TH per monocyte, without increasing the total numbers of monocytes. These results revealed that TNF{delta} could potentially modify monocytic TH production and serve a regulatory role in peripheral immune function. The development and application of a highly sensitive assay to quantify TH in both human and animal cells will provide a novel tool for further investigating possible PD immune regulatory pathways between brain and periphery.

neuroscience↗

bioRxiv2021

A study of the neuronal injury biomarkers pNF-H and UCHL1 in serum, CSF and urine in a cohort of thoracic endovascular aortic repair (TEVAR) patients.

A collection of longitudinal serum, cerebrospinal fluid (CSF) and urine samples were collected from a cohort of 50 patients undergoing thoracic endovascular aortic repairs (TEVAR). Samples were taken multiple time per day out to 5 days post operation and were probed with novel electrochemiluminescent assays specific for the phosphorylated axonal form of the major neurofilament subunit NF-H (pNF-H) and for ubiquitin C-terminal hydrolase 1 (UCHL1). Control blood samples showed small signals for pNF-H and in some cases rather larger signals for UCHL1. The presence of UCHL1 in these control blood samples was convincingly verified by western blotting with multiple well characterized UCHL1 antibodies and by mass spectroscopy. Elevated levels of both pNF-H and UCHL1 in blood and CSF in recovering TEVAR patients were associated with poorer outcomes. In particular release of UCHL1 into blood over several hours following TEVAR and peaking at any time over 1 ng/ml was a very strong predictor of patient death and was associated with renal failure and spinal cord ischemia (SCI). An unexpected finding was that high levels of UCHL1 were detected in certain urine samples, again in association with SCI, renal failure and poor patient outcome. We also present epitope mapping data on the UCHL1 monoclonal antibodies used including data on the widely used commercially available MCA-BH7. These studies suggest that measurement of the levels of both proteins in the blood, CSF and urine of TEVAR patients may be of clinical utility. However this study also raises questions about the origin and significance of UCHL1 both in control blood, in patient blood samples and in urine.

neuroscience↗

bioRxiv2020

Traumatic stress history interacts with chronic peripheral inflammation to alter mitochondrial function of synaptosomes in a sex-specific manner

BackgroundRepeated exposures to chronic stress can lead to long lasting negative behavioral and metabolic outcomes. Here, we aim to determine the impact of chronic stress and chronic low-level inflammation on behavior and synaptosomal metabolism. MethodsMale (n = 31) and female (n = 32) C57Bl/6 mice underwent chronic repeated predation stress or daily handling for two rounds of 15 consecutive days of exposure during the adolescent and early adult timeframes. Subsequently, mice were exposed to repeated lipopolysaccharide (LPS; 7.5 x 105 EU/kg) or saline injections every third day for eight weeks. Exploratory and social behaviors were assessed in the open field and social interaction tests prior to examination of learning and memory with the Barnes Maze. Mitochondrial function and morphology were assessed in synaptosomes post-mortem. In addition, expression of TNF-, IL-1{beta}, and ROMO1 were examined in the hippocampus and prefrontal cortex. Circulating pro- and anti-inflammatory cytokines in the periphery were assessed following the first and last LPS injection as well as at the time of tissue collection. Circulating ROMO1 was assessed in terminal samples. ResultsExposure to repeated predatory stress increased time spent in the corners of the open field, suggestive of anxiety-like behavior, in both males and females. There were no significant group differences in the social interaction test and minimal effects were evident in the Barnes maze. A history of chronic stress interacted with chronic LPS in male mice to lead to a deficit in synaptosomal respiration. Female mice were more sensitive to both chronic stress and chronic LPS such that either a history of chronic stress or a history of chronic LPS was sufficient to disrupt synaptosomal respiration in females. Both stress and chronic LPS were sufficient to increase inflammation and reactive oxygen in males in the periphery and centrally. Females had increased markers of peripheral inflammation following acute LPS but no evidence of peripheral or central increases in inflammatory factors or reactive oxygen following chronic exposures. ConclusionCollectively, these data suggest that while metrics of inflammation and reactive oxygen are disrupted in males following chronic stress and chronic LPS, only the combined condition is sufficient to alter synaptosomal respiration. Conversely, although evidence of chronic inflammation or chronic elevation in reactive oxygen is absent, females demonstrate profound shifts in synaptosomal mitochondrial function with either a history of chronic stress or a history of chronic inflammation. These data highlight that differential mechanisms are likely in play between the sexes and suggest that female sensitivity to neurogenerative conditions may be precipitated by influence of life experiences on mitochondrial function in the synapses.

neuroscience↗

bioRxiv2019

Accumulation of Rare Coding Variants in Genes Implicated in Risk of Human Cleft Lip with or without Cleft Palate

Cleft lip with/without cleft palate (CLP) is a common craniofacial malformation with complex etiologies, reflecting both genetic and environmental factors. Most of the suspected genetic risk for CLP has yet to be identified. To further classify risk loci and estimate the contribution of rare variants, we sequenced the exons in 49 candidate genes in 323 CLP cases and 211 non-malformed controls. Our findings indicated that rare, protein-altering variants displayed markedly higher burdens in CLP cases at relevant loci. First, putative loss-of-function mutations (nonsense, frameshift) were significantly enriched among cases: 13 of 323 cases (~4%) harbored such alleles within these 49 genes, versus one such change in controls (p = 0.01). Second, in gene-level analyses, the burden of rare alleles showed greater case-association for several genes previously implicated in cleft risk. For example, BHMT displayed a 10-fold increase in protein-altering variants in CLP cases (p = 0.03), including multiple case occurrences of a rare frameshift mutation (K400fs). Other loci with greater rare, coding allele burdens in cases were in signaling pathways relevant to craniofacial development (WNT9B, BMP4, BMPR1B) as well as the methionine cycle (MTRR). We conclude that rare coding variants may confer risk for isolated CLP.

genetics↗