Somatic mosaicism in neuronal precursor cells mediated by L1 retrotransposition

Revealing the mechanisms for neuronal somatic diversification remains a central challenge for understanding individual differences in brain organization and function. Here we show that an engineered human LINE-1 (for long interspersed nuclear element-1; also known as L1) element can retrotranspose in neuronal precursors derived from rat hippocampus neural stem cells. The resulting retrotransposition events can alter the expression of neuronal genes, which, in turn, can influence neuronal cell fate in vitro. We further show that retrotransposition of a human L1 in transgenic mice results in neuronal somatic mosaicism. The molecular mechanism of action is probably mediated through Sox2, because a decrease in Sox2 expression during the early stages of neuronal differentiation is correlated with increases in both L1 transcription and retrotransposition. Our data therefore indicate that neuronal genomes might not be static, but some might be mosaic because of de novo L1 retrotransposition events.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/62714/1/nature03663.pd

Numt-Mediated Double-Strand Break Repair Mitigates Deletions during Primate Genome Evolution

Non-homologous end joining (NHEJ) is the major mechanism of double-strand break repair (DSBR) in mammalian cells. NHEJ has traditionally been inferred from experimental systems involving induced double strand breaks (DSBs). Whether or not the spectrum of repair events observed in experimental NHEJ reflects the repair of natural breaks by NHEJ during chromosomal evolution is an unresolved issue. In primate phylogeny, nuclear DNA sequences of mitochondrial origin, numts, are inserted into naturally occurring chromosomal breaks via NHEJ. Thus, numt integration sites harbor evidence for the mechanisms that act on the genome over evolutionary timescales. We have identified 35 and 55 lineage-specific numts in the human and chimpanzee genomes, respectively, using the rhesus monkey genome as an outgroup. One hundred and fifty two numt-chromosome fusion points were classified based on their repair patterns. Repair involving microhomology and repair leading to nucleotide additions were detected. These repair patterns are within the experimentally determined spectrum of classical NHEJ, suggesting that information from experimental systems is representative of broader genetic loci and end configurations. However, in incompatible DSBR events, small deletions always occur, whereas in 54% of numt integration events examined, no deletions were detected. Numts show a statistically significant reduction in deletion frequency, even in comparison to DSBR involving filler DNA. Therefore, numts show a unique mechanism of integration via NHEJ. Since the deletion frequency during numt insertion is low, native overhangs of chromosome breaks are preserved, allowing us to determine that 24% of the analyzed breaks are cohesive with overhangs of up to 11 bases. These data represent, to the best of our knowledge, the most comprehensive description of the structure of naturally occurring DSBs. We suggest a model in which the sealing of DSBs by numts, and probably by other filler DNA, prevents nuclear processing of DSBs that could result in deleterious repair

The RNA Polymerase Dictates ORF1 Requirement and Timing of LINE and SINE Retrotransposition

Mobile elements comprise close to one half of the mass of the human genome. Only LINE-1 (L1), an autonomous non-Long Terminal Repeat (LTR) retrotransposon, and its non-autonomous partners—such as the retropseudogenes, SVA, and the SINE, Alu—are currently active human retroelements. Experimental evidence shows that Alu retrotransposition depends on L1 ORF2 protein, which has led to the presumption that LINEs and SINEs share the same basic insertional mechanism. Our data demonstrate clear differences in the time required to generate insertions between marked Alu and L1 elements. In our tissue culture system, the process of L1 insertion requires close to 48 hours. In contrast to the RNA pol II-driven L1, we find that pol III transcribed elements (Alu, the rodent SINE B2, and the 7SL, U6 and hY sequences) can generate inserts within 24 hours or less. Our analyses demonstrate that the observed retrotransposition timing does not dictate insertion rate and is independent of the type of reporter cassette utilized. The additional time requirement by L1 cannot be directly attributed to differences in transcription, transcript length, splicing processes, ORF2 protein production, or the ability of functional ORF2p to reach the nucleus. However, the insertion rate of a marked Alu transcript drastically drops when driven by an RNA pol II promoter (CMV) and the retrotransposition timing parallels that of L1. Furthermore, the “pol II Alu transcript” behaves like the processed pseudogenes in our retrotransposition assay, requiring supplementation with L1 ORF1p in addition to ORF2p. We postulate that the observed differences in retrotransposition kinetics of these elements are dictated by the type of RNA polymerase generating the transcript. We present a model that highlights the critical differences of LINE and SINE transcripts that likely define their retrotransposition timing

Characterization of LINE-1 Ribonucleoprotein Particles

The average human genome contains a small cohort of active L1 retrotransposons that encode two proteins (ORF1p and ORF2p) required for their mobility (i.e., retrotransposition). Prior studies demonstrated that human ORF1p, L1 RNA, and an ORF2p-encoded reverse transcriptase activity are present in ribonucleoprotein (RNP) complexes. However, the inability to physically detect ORF2p from engineered human L1 constructs has remained a technical challenge in the field. Here, we have employed an epitope/RNA tagging strategy with engineered human L1 retrotransposons to identify ORF1p, ORF2p, and L1 RNA in a RNP complex. We next used this system to assess how mutations in ORF1p and/or ORF2p impact RNP formation. Importantly, we demonstrate that mutations in the coiled-coil domain and RNA recognition motif of ORF1p, as well as the cysteine-rich domain of ORF2p, reduce the levels of ORF1p and/or ORF2p in L1 RNPs. Finally, we used this tagging strategy to localize the L1–encoded proteins and L1 RNA to cytoplasmic foci that often were associated with stress granules. Thus, we conclude that a precise interplay among ORF1p, ORF2p, and L1 RNA is critical for L1 RNP assembly, function, and L1 retrotransposition

Adenosine stress CMR T1-mapping detects early microvascular dysfunction in patients with type 2 diabetes mellitus without obstructive coronary artery disease

Background Type 2 diabetes mellitus (T2DM) is associated with coronary microvascular dysfunction in the absence of obstructive coronary artery disease (CAD). Cardiovascular magnetic resonance (CMR) T1-mapping at rest and during adenosine stress can assess coronary vascular reactivity. We hypothesised that the non-contrast T1 response to vasodilator stress will be altered in patients with T2DM without CAD compared to controls due to coronary microvascular dysfunction. Methods Thirty-one patients with T2DM and sixteen matched healthy controls underwent CMR (3 T) for cine, rest and adenosine stress non-contrast T1-mapping (ShMOLLI), first-pass perfusion and late gadolinium enhancement (LGE) imaging. Significant CAD (>50% coronary luminal stenosis) was excluded in all patients by coronary computed tomographic angiography. Results All subjects had normal left ventricular (LV) ejection and LV mass index, with no LGE. Myocardial perfusion reserve index (MPRI) was lower in T2DM than in controls (1.60 ± 0.44 vs 2.01 ± 0.42; p = 0.008). There was no difference in rest native T1 values (p = 0.59). During adenosine stress, T1 values increased significantly in both T2DM patients (from 1196 ± 32 ms to 1244 ± 44 ms, p < 0.001) and controls (from 1194 ± 26 ms to 1273 ± 44 ms, p < 0.001). T2DM patients showed blunted relative stress non-contrast T1 response (T2DM: ΔT1 = 4.1 ± 2.9% vs. controls: ΔT1 = 6.6 ± 2.6%, p = 0.007) due to a blunted maximal T1 during adenosine stress (T2DM 1244 ± 44 ms vs. controls 1273 ± 44 ms, p = 0.045). Conclusions Patients with well controlled T2DM, even in the absence of arterial hypertension and significant CAD, exhibit blunted maximal non-contrast T1 response during adenosine vasodilatory stress, likely reflecting coronary microvascular dysfunction. Adenosine stress and rest T1 mapping can detect subclinical abnormalities of the coronary microvasculature, without the need for gadolinium contrast agents. CMR may identify early features of the diabetic heart phenotype and subclinical cardiac risk markers in patients with T2DM, providing an opportunity for early therapeutic intervention

Evaluation of appendicitis risk prediction models in adults with suspected appendicitis

Background Appendicitis is the most common general surgical emergency worldwide, but its diagnosis remains challenging. The aim of this study was to determine whether existing risk prediction models can reliably identify patients presenting to hospital in the UK with acute right iliac fossa (RIF) pain who are at low risk of appendicitis. Methods A systematic search was completed to identify all existing appendicitis risk prediction models. Models were validated using UK data from an international prospective cohort study that captured consecutive patients aged 16–45 years presenting to hospital with acute RIF in March to June 2017. The main outcome was best achievable model specificity (proportion of patients who did not have appendicitis correctly classified as low risk) whilst maintaining a failure rate below 5 per cent (proportion of patients identified as low risk who actually had appendicitis). Results Some 5345 patients across 154 UK hospitals were identified, of which two‐thirds (3613 of 5345, 67·6 per cent) were women. Women were more than twice as likely to undergo surgery with removal of a histologically normal appendix (272 of 964, 28·2 per cent) than men (120 of 993, 12·1 per cent) (relative risk 2·33, 95 per cent c.i. 1·92 to 2·84; P < 0·001). Of 15 validated risk prediction models, the Adult Appendicitis Score performed best (cut‐off score 8 or less, specificity 63·1 per cent, failure rate 3·7 per cent). The Appendicitis Inflammatory Response Score performed best for men (cut‐off score 2 or less, specificity 24·7 per cent, failure rate 2·4 per cent). Conclusion Women in the UK had a disproportionate risk of admission without surgical intervention and had high rates of normal appendicectomy. Risk prediction models to support shared decision‐making by identifying adults in the UK at low risk of appendicitis were identified

Assessing the practicality of a novel heart failure therapeutic score (QUAD Score) for drug optimisation in heart failure with a reduced ejection fraction

Abstract Introduction The 4 pillars of drug treatment for heart failure with a reduced ejection fraction (HFrEF) [Angiotensin-converting enzyme inhibitors (ACEi)/Angiotensin receptor–neprilysin inhibitors (ARNi), β-blockers (BB), Mineralocorticoid receptor antagonists (MRA), and Sodium-glucose cotransporter 2 inhibitors (SGLT2i)], are recommended by international HF guidelines to improve patient outcomes. Treatment inertia remains a challenge with non-prescription or sub-optimal dosing of these therapies. We developed a pragmatic HFrEF treatment score (QUAD score) (Figure 1) to prompt initiation and/or titration of sub-optimally dosed foundational therapies. Patients are categorized as poor (<8), good (8-14), and excellent (15-24) based on the dose and number of foundational drugs prescribed. This study aims to assess the practical utility of the QUAD score and its influence on treatment optimization. Methods Unwarned prescribing HCPs were approached consecutively in person, within clinical settings. Participants were told the questions were part of a research project and they provided verbal consent. Responses were anonymous and no patients were involved; therefore, verbal consent was considered proportionate and reasonable. HCPs were given an information sheet describing the QUAD score (Figure 1). They were then presented with 3 hypothetical clinical scenarios of haemodynamically stable and uncomplicated HFrEF patients with uncalculated poor, good and excellent QUAD scores. They were asked to calculate the QUAD score and if they proposed any changes to treatment. Time was recorded using a stopwatch and proposed changes were recorded. They were then invited to answer a series of follow-on questions. Results Between September 2023 and February 2024, there were 70 respondents. 71% (50) were doctors and 29% (20) allied HCPs. 83% (58) placed patients in the correct treatment grade in at least one scenario. 62.9% (44) did so for all three scenarios (Figure 2) with numerically more HCPs compared to doctors (80% vs 56% p=0.06).The median (IQR) time in seconds required to calculate the QUAD score for clinical scenarios 1,2 and 3 was 158 (100-234), 86 (54-122) and 83 (53-119) respectively. In response to the calculated QUAD score, 94.3% (66), recommended drug treatment optimisations. On a 5-point Likert scale (1 -poor, 5- excellent), 77.1% of participants rated the QUAD score ≥3 for ease of use, and 88.6% rated ≥3 as an incentive for treatment optimization. Conclusion We demonstrated the rapid clinical usability of the QUAD Score in a cohort of unselected prescribing HCPs, with a decremental calculation time with each presented clinical scenario. HCPs were also motivated to achieve further HF drug therapy optimization based on the calculated score. We conclude that the QUAD Score as a HFrEF drug therapy score may have a role in addressing treatment inertia however, it needs validation against clinically relevant outcomes in a larger cohort of HFrEF patients.</p