Biology subjects

Tan, T. Y.

Publications and source records attributed to Tan, T. Y..

7 recordsLinked to original sources

bioRxiv2025

Minute amounts of helicase-deficient truncated RECQL4 are sufficient for DNA replication.

RECQL4 is a member of the RecQ family of helicases, playing essential roles in DNA replication and maintaining genome integrity. Mutations in RECQL4 are linked to severe human diseases, including Rothmund-Thomson Syndrome, RAPIDALINO Syndrome, and Baller-Gerold Syndrome. However, we still do not fully understand its functions and genetic interactions. The role of the ATP-dependent helicase activity in RECQL4 remains controversial. To understand RECQL4s functions further, we conducted a genome-wide forward genetic screen using murine models that closely mimic the RECQL4 mutations found in patients with Rothmund-Thomson syndrome. Our goal was to identify loss-of-function alleles that could rescue the proliferation and viability defects associated with RECQL4 mutation. From our screening we identified the loss of KLHDC3, a substrate-binding subunit of the Cullin-RING ligase (CRL) E3, as the most significant rescue allele. KLHDC3 facilitates the ubiquitin-mediated destruction of proteins with specific C-terminal degron motifs. Its loss normalized cell proliferation and DNA replication rates in cells with mutated RECQL4. Further analysis revealed that the loss of KLHDC3 led to the stabilization of minute levels of a truncated RECQL4 protein. This RECQL4 fragment contained a neo-degron sequence specific for KLHDC3, formed after Cre-mediated recombination of the Recql4fl allele. Although this rescue mechanism does not apply to human RECQL4 mutations, it shows that very low chromatin-bound levels of a truncated RECQL4 protein--comprising only the N-terminal 480 amino acids, including its Sld2-like domain but lacking the ATP-dependent helicase domain and the entire C-terminal portion--are sufficient to support DNA replication in mammalian cells. These results demonstrate that the ATPase activity and helicase domain of RECQL4 are not essential for DNA replication in mammals. Furthermore, our findings suggest that there are unlikely to be monogenic loss-of-function alleles that can rescue RECQL4 mutations. This demonstrates that RECQL4 is an essential and non-redundant regulator of DNA replication and cell viability and that this activity does not require the ATP dependent helicase activity.

cell biology↗

bioRxiv2022

Peritoneal autoantibody landscape in endometriosis

Women with endometriosis have a profound association with autoimmunity. An excess of autoantigens in the peritoneal cavity resulting from retrograde menstruation could lead to inflammation and pathologic autoimmunity. Using a native-conformation protein array, proteome-wide analysis of autoantibodies (AAbs) against 1623 proteins were profiled in peritoneal fluids (PF) of 25 women with endometriosis and 25 endometriosis-negative women. 46% of endometriotic women have five or more AAbs. Diverse cognate autoantigens were identified and corresponding AAbs against proteins involved in implantation, B-cell activation/development, and aberrant migration and mitogenicity. AAbs recognizing tumour suppressor protein p53 were the most frequent at 35% and were targeted against native and citrullinated p53 forms. Further, unsupervised hierarchical clustering and integrative pathway analysis, we observed clusters of endometriosis-associated infertile women with 60% positive for two or more AAbs which are involved in PDGF, TGF-{beta}, RAC1/PAK1/p38/MMP2 signaling, LAT2/NTAL/LAB-mediated calcium mobilisation and integrin-mediated cell adhesion. Together, our data identifies peritoneal autoimmunity in a significant subset of women with endometriosis, with diverse impact on infertility and disease pathophysiology.

immunology↗

bioRxiv2020

Supporting Student Learning and Experiences in the Lab: (How) Should We Design Their Groups?

Undergraduate science students spend a substantial amount of time working in their laboratory groups, and instructors want to make evidence-based decisions on how to best set up these groups. Despite several studies on group composition, the evidence appears to be quite context-specific, and very little has been published about lab groups. Further, many studies focus solely on conceptual learning; however, the lab is an important venue for also supporting non-content outcomes such as confidence, process skills, team skills, and attitudes. Thus, in our introductory course on molecules, cells, and physiology we were interested in the impact of group composition, on a spread of student outcomes. Students were either placed into groups by the instructor, or self-selected into groups. To assess the impact of group composition on student outcome, we collected pre/post data from >500 students over 2 semesters. Our measures assess conceptual knowledge, confidence in lab skills, attitudes toward group learning, lab grades, gender, year of study, and (via open-ended questions) student perspectives. Using a multiple regression approach, we established models that predict student outcomes based on their individual attributes and on their lab group attributes. Surprisingly, the hetero/homogeneity of the initial group, and whether the groups were student- or instructor-selected, did not affect student outcomes in these models. Further MANCOVA analysis demonstrated that student interaction outside of the lab time was the strongest predictor of positive student attitudes toward group learning. Student perspectives on group formation are mixed, and suggest that a simple and flexible choice approach may best support our students. Overall, these findings have clear implications for our course design and instructional choices: we should focus our efforts to promote positive student interactions, rather than worrying about initial composition.

scientific communication and education↗

bioRxiv2020

The chromatin remodeler ISWI acts during Drosophila development to regulate adult sleep

Sleep disruptions are among the most commonly-reported symptoms across neurodevelopmental disorders (NDDs), but mechanisms linking brain development to normal sleep are largely unknown. From a Drosophila screen of human NDD-associated risk genes, we identified the chromatin remodeler Imitation SWItch/SNF (ISWI) to be required for adult fly sleep. Loss of ISWI also results in disrupted circadian rhythms, memory, and social behavior, but ISWI acts in different cells and during distinct developmental times to affect each of these adult behaviors. Specifically, ISWI expression in type I neuroblasts is required for both adult sleep and formation of a learning-associated brain region. Expression in flies of the human ISWI homologs SMARCA1 and SMARCA5 differentially rescue adult phenotypes. We propose that sleep deficits are a primary phenotype of early developmental origin in NDDs, and point towards chromatin remodeling machinery as critical for sleep circuit formation.

neuroscience↗

bioRxiv2019

Metabolic stress is a primary pathogenic event in transgenic Caenorhabditis elegans expressing neuronal human amyloid-β

Alzheimers disease (AD) is the most common neurodegenerative disease affecting the elderly worldwide. Mitochondrial dysfunction has been proposed as a key event in the etiology of AD. We have previously modeled amyloid-beta (A{beta})-induced mitochondrial dysfunction in a transgenic Caenorhabditis elegans strain by expressing human A{beta} peptide specifically in neurons (GRU102). Here, we focus on a deeper analysis of these metabolic changes associated with A{beta}-induced mitochondrial dysfunction. Integrating metabolomics, transcriptomics, biochemical studies and computational modeling, we identify alterations in Tricarboxylic Acid (TCA) cycle metabolism following even low-level A{beta} expression. In particular, GRU102 show reduced activity of a rate-limiting TCA cycle enzyme, alpha-ketoglutarate dehydrogenase. These defects are associated with elevation of protein carbonyl content specifically in mitochondria. Importantly, metabolic failure occurs before any significant increase in global protein aggregate is detectable. Treatment with an antidiabetes drug, Metformin, reverses A{beta}-induced metabolic defects, reduces protein aggregation and normalizes the lifespan of GRU102. Our results point to metabolic dysfunction as an early and causative event in AD pathology and a promising target for intervention.

neuroscience↗

bioRxiv2019

Pooled-parent exome sequencing to prioritise de novo variants in genetic disease

In the clinical setting, exome sequencing has become standard-of-care in diagnosing rare genetic disorders, however many patients remain unsolved. Trio sequencing has been demonstrated to produce a higher diagnostic yield than singleton (proband-only) sequencing. Parental sequencing is especially useful when a disease is suspected to be caused by a de novo variant in the proband, because parental data provide a strong filter for the majority of variants that are shared by the proband and their parents. However the additional cost of sequencing the parents makes the trio strategy uneconomical for many clinical situations. With two thirds of the sequencing budget being spent on parents, these are funds that could be used to sequence more probands. For this reason many clinics are reluctant to sequence parents.\n\nHere we propose a pooled-parent strategy for exome sequencing of individuals with likely de novo disease. In this strategy, DNA from all the parents of a cohort of unrelated probands is pooled together into a single exome capture and sequencing run. Variants called in the proband can then be filtered if they are also found in the parent pool, resulting in a shorter list of prioritised variants. To evaluate the pooled-parent strategy we performed a series of simulations by combining reads from individual exomes to imitate sample pooling. We assessed the recall and false positive rate and investigated the trade-off between pool size and recall rate. We compared the performance of GATK HaplotypeCaller individual and joint calling, and FreeBayes to genotype pooled samples. Finally, we applied a pooled-parent strategy to a set of real unsolved cases and showed that the parent pool is a powerful filter that is complementary to other commonly used variant filters such as population variant frequencies.

bioinformatics↗

bioRxiv2018

A meta-analysis of the diagnostic sensitivity and clinical utility of genome sequencing, exome sequencing and chromosomal microarray in children with suspected genetic diseases

IMPORTANCEGenetic diseases are a leading cause of childhood mortality. Whole genome sequencing (WGS) and whole exome sequencing (WES) are relatively new methods for diagnosing genetic diseases.\n\nOBJECTIVESCompare the diagnostic sensitivity (rate of causative, pathogenic or likely pathogenic genotypes in known disease genes) and rate of clinical utility (proportion in whom medical or surgical management was changed by diagnosis) of WGS, WES, and chromosomal microarrays (CMA) in children with suspected genetic diseases.\n\nDATA SOURCES AND STUDY SELECTIONSystematic review of the literature (January 2011 - August 2017) for studies of diagnostic sensitivity and/or clinical utility of WGS, WES, and/or CMA in children with suspected genetic diseases. 2% of identified studies met selection criteria.\n\nDATA EXTRACTION AND SYNTHESISTwo investigators extracted data independently following MOOSE/PRISMA guidelines.\n\nMAIN OUTCOMES AND MEASURESPooled rates and 95% Cl were estimated with a random-effects model. Metaanalysis of the rate of diagnosis was based on test type, family structure, and site of testing.\n\nRESULTSIn 36 observational series and one randomized control trial, comprising 20,068 children, the diagnostic sensitivity of WGS (0.41, 95% Cl 0.34-0.48, I2=44%) and WES (0.35, 95% Cl 0.31-0.39, I2=85%) were qualitatively greater than CMA (0.10, 95% Cl 0.08-0.12, I2=81%). Subgroup meta-analyses showed that the diagnostic sensitivity of WGS was significantly greater than CMA in studies published in 2017 (P<.0001, I2=13% and I2=40%, respectively), and the diagnostic sensitivity of WES was significantly greater than CMA in studies featuring within-cohort comparisons (P<001, I2=36%). Evidence for a significant difference in the diagnostic sensitivity of WGS and WES was lacking. In studies featuring within-cohort comparisons of singleton and trio WGS/WES, the likelihood of diagnosis was significantly greater for trios (odds ratio 2.04, 95% Cl 1.62-2.56, I2=12%; P<.0001). The diagnostic sensitivity of WGS/WES with hospital-based interpretation (0.41, 95% Cl 0.38-0.45, I2=50%) was qualitatively higher than that of reference laboratories (0.28, 95% Cl 0.24-0.32, I2=81%); this difference was significant in meta-analysis of studies published in 2017 (P=.004, I2=34% and I2=26%, respectively). The rates of clinical utility of WGS (0.27, 95% Cl 0.17-0.40, I2=54%) and WES (0.18, 95% Cl 0.13-0.24, I2-77%) were higher than CMA (0.06, 95% Cl 0.05-0.07, I2=42%); this difference was significant in meta-analysis of WGS vs CMA (P<.0001).\n\nCONCLUSIONS AND RELEVANCEIn children with suspected genetic diseases, the diagnostic sensitivity and rate of clinical utility of WGS/WES were greater than CMA. Subgroups with higher WGS/WES diagnostic sensitivity were trios and those receiving hospital-based interpretation. WGS/WES should be considered a first-line genomic test for children with suspected genetic diseases.\n\nKey PointsO_ST_ABSQuestionC_ST_ABSWhat is the relative diagnostic sensitivity and clinical utility of different genome tests in children with suspected genetic diseases?\n\nFindingsWhole genome sequencing had greater diagnostic sensitivity and clinical utility than chromosomal microarrays. Testing parent-child trios had greater diagnostic sensitivity than proband singletons. Hospital-based testing had greater diagnostic sensitivity than reference laboratories.\n\nMeaningTrio genomic sequencing is the most sensitive diagnostic test for children with suspected genetic diseases.

genomics↗