PRDM9 is a major specifier of human meiotic recombination hotspots, probably via binding of its zinc-finger repeat array to a DNA sequence motif associated with hotspots. However, our view of PRDM9 regulation, in terms of motifs defined and hotspots studied, has a strong bias toward the PRDM9 A variant particularly common in Europeans. We show that population diversity can reveal a second class of hotspots specifically activated by PRDM9 variants common in Africans but rare in Europeans. These Africanenhanced hotspots nevertheless share very similar properties with their counterparts activated by the A variant. The specificity of hotspot activation is such that individuals with differing PRDM9 genotypes, even within the same population, can use substantially if not completely different sets of hotspots. Each African-enhanced hotspot is activated by a distinct spectrum of PRDM9 variants, despite the fact that all are predicted to bind the same sequence motif. This differential activation points to complex interactions between the zinc-finger array and hotspots and identifies features of the array that might be important in controlling hotspot activity.eiotic recombination is fundamentally important in ensuring correct chromosome disjunction at meiosis and in reshuffling haplotypes between generations, substantially increasing haplotype diversity within a population. Most recombination events in the human genome are clustered into narrow hotspots that can be identified indirectly from patterns of linkage disequilibrium (LD hotspots) (1), or directly through high-resolution linkage analysis in pedigrees (2) or by sperm typing (3). Genomewide comparison of LD hotspots has identified a sequence motif CCNCCNTNNCCNC associated with 40% of these hotspots; this motif appears to influence the initiation of meiotic recombination, because SNPs that disrupt the motif can down-regulate recombination (4).Recently, the meiosis-specific protein PRDM9 has been identified as a major specifier of hotspots in the human and mouse genome (5-7). PRDM9 contains a SET domain that might be responsible for activating hotspots by chromatin remodelling (8), plus a C-terminal tandem-repeat zinc-finger (ZnF) array encoded by a variable minisatellite. Evidence that PRDM9 regulates hotspots comes from the finding that the common European variant A has a ZnF array that binds, at least in vitro, to the 13-mer hotspot motif shared by many LD hotspots identified in Europeans (5, 6). Furthermore, association analyses in Hutterites (5) and Icelanders (2) have shown that individuals with variant non-A PRDM9 alleles can show genome-wide shifts in hotspot usage. These shifts suggest that ZnF variants that should not bind the PRDM9 A motif might trigger the appearance of new sets of hotspots (5), although it is possible that some of these shifts reflect additional hotspot-specification systems that only become manifest as the dosage of the PRDM9 A variant is reduced. The Icelandic study highlighted the PRDM9 C variant and its associated predicted motif CC...

SummaryBackgroundCerebral microbleeds are a potential neuroimaging biomarker of cerebral small vessel diseases that are prone to intracranial bleeding. We aimed to determine whether presence of cerebral microbleeds can identify patients at high risk of symptomatic intracranial haemorrhage when anticoagulated for atrial fibrillation after recent ischaemic stroke or transient ischaemic attack.MethodsOur observational, multicentre, prospective inception cohort study recruited adults aged 18 years or older from 79 hospitals in the UK and one in the Netherlands with atrial fibrillation and recent acute ischaemic stroke or transient ischaemic attack, treated with a vitamin K antagonist or direct oral anticoagulant, and followed up for 24 months using general practitioner and patient postal questionnaires, telephone interviews, hospital visits, and National Health Service digital data on hospital admissions or death. We excluded patients if they could not undergo MRI, had a definite contraindication to anticoagulation, or had previously received therapeutic anticoagulation. The primary outcome was symptomatic intracranial haemorrhage occurring at any time before the final follow-up at 24 months. The log-rank test was used to compare rates of intracranial haemorrhage between those with and without cerebral microbleeds. We developed two prediction models using Cox regression: first, including all predictors associated with intracranial haemorrhage at the 20% level in univariable analysis; and second, including cerebral microbleed presence and HAS-BLED score. We then compared these with the HAS-BLED score alone. This study is registered with ClinicalTrials.gov, number NCT02513316.FindingsBetween Aug 4, 2011, and July 31, 2015, we recruited 1490 participants of whom follow-up data were available for 1447 (97%), over a mean period of 850 days (SD 373; 3366 patient-years). The symptomatic intracranial haemorrhage rate in patients with cerebral microbleeds was 9·8 per 1000 patient-years (95% CI 4·0–20·3) compared with 2·6 per 1000 patient-years (95% CI 1·1–5·4) in those without cerebral microbleeds (adjusted hazard ratio 3·67, 95% CI 1·27–10·60). Compared with the HAS-BLED score alone (C-index 0·41, 95% CI 0·29–0·53), models including cerebral microbleeds and HAS-BLED (0·66, 0·53–0·80) and cerebral microbleeds, diabetes, anticoagulant type, and HAS-BLED (0·74, 0·60–0·88) predicted symptomatic intracranial haemorrhage significantly better (difference in C-index 0·25, 95% CI 0·07–0·43, p=0·0065; and 0·33, 0·14–0·51, p=0·00059, respectively).InterpretationIn patients with atrial fibrillation anticoagulated after recent ischaemic stroke or transient ischaemic attack, cerebral microbleed presence is independently associated with symptomatic intracranial haemorrhage risk and could be used to inform anticoagulation decisions. Large-scale collaborative observational cohort analyses are needed to refine and validate intracranial haemorrhage risk scores incorporating cerebral microbleeds to identify patients at risk of net harm from ora...

Huntington's disease is a fatal neurodegenerative disorder caused by a CAG repeat expansion encoding a polyglutamine tract in the huntingtin (Htt) protein. Here we report a genome-wide overexpression suppressor screen in which we identified 317 ORFs that ameliorate the toxicity of a mutant Htt fragment in yeast and that have roles in diverse cellular processes, including mitochondrial import and copper metabolism. Two of these suppressors encode glutathione peroxidases (GPxs), which are conserved antioxidant enzymes that catalyze the reduction of hydrogen peroxide and lipid hydroperoxides. Using genetic and pharmacological approaches in yeast, mammalian cells and Drosophila, we found that GPx activity robustly ameliorates Huntington's disease-relevant metrics and is more protective than other antioxidant approaches tested here. Notably, we found that GPx activity, unlike many antioxidant treatments, does not inhibit autophagy, which is an important mechanism for clearing mutant Htt. Because previous clinical trials have indicated that GPx mimetics are well tolerated in humans, this study may have important implications for treating Huntington's disease.

Huntington disease (HD) is a neurodegenerative disorder caused by the expansion of a polyglutamine tract in the huntingtin (htt) protein. To uncover candidate therapeutic targets and networks involved in pathogenesis, we integrated gene expression profiling and functional genetic screening to identify genes critical for mutant htt toxicity in yeast. Using mRNA profiling, we have identified genes differentially expressed in wild-type yeast in response to mutant htt toxicity as well as in three toxicity suppressor strains: bna4Δ, mbf1Δ, and ume1Δ. BNA4 encodes the yeast homolog of kynurenine 3-monooxygenase, a promising drug target for HD. Intriguingly, despite playing diverse cellular roles, these three suppressors share common differentially expressed genes involved in stress response, translation elongation, and mitochondrial transport. We then systematically tested the ability of the differentially expressed genes to suppress mutant htt toxicity when overexpressed and have thereby identified 12 novel suppressors, including genes that play a role in stress response, Golgi to endosome transport, and rRNA processing. Integrating the mRNA profiling data and the genetic screening data, we have generated a robust network that shows enrichment in genes involved in rRNA processing and ribosome biogenesis. Strikingly, these observations implicate dysfunction of translation in the pathology of HD. Recent work has shown that regulation of translation is critical for life span extension in Drosophila and that manipulation of this process is protective in Parkinson disease models. In total, these observations suggest that pharmacological manipulation of translation may have therapeutic value in HD.

A 5.5-year-old child with nephrotic syndrome was treated with cyclophosphamide. After 9 weeks of therapy she developed jaundice and abnormal liver function tests. No infective aetiology was found and the abnormal liver function tests resolved within 5 weeks of discontinuing cyclophosphamide. Cyclophosphamide has rarely been reported to cause liver dysfunction, but not in children treated for nephrotic syndrome, and paediatricians should therefore be aware of its potential for inducing reversible hepatic dysfunction.

A 5.5-year-old child with nephrotic syndrome was treated with cyclophosphamide. After 9 weeks of therapy she developed jaundice and abnormal liver function tests. No infective aetiology was found and the abnormal liver function tests resolved within 5 weeks of discontinuing cyclophosphamide. Cyclophosphamide has rarely been reported to cause liver dysfunction, but not in children treated for nephrotic syndrome, and paediatricians should therefore be aware of its potential for inducing reversible hepatic dysfunction.

AimsVariable rate intravenous insulin infusion (VRIII) is used perioperatively to maintain normoglycaemia in patients with diabetes who are undergoing surgery. The aims of this project were as follows: (1) to audit the extent to which perioperative prescribing of VRIII for diabetic vascular surgery inpatients at our hospital meets established standards and (2) to use the results of the audit to guide improvement in the quality and safety of prescribing practices and reduce VRIII overuse.MethodsVascular surgery inpatients who had perioperative VRIII were included in the audit. Baseline data were collected consecutively from September to November 2021. There were three main interventions: a VRIII Prescribing Checklist, education of junior doctors and ward staff, and electronic prescribing system updates. Postintervention and reaudit data were collected consecutively from March to June 2022.ResultsThe number of VRIII prescriptions totalled 27 in preintervention, 18 in postintervention and 26 in reaudit periods. Prescribers used the ‘refer to paper chart’ safety check more frequently postintervention (67%) and on reaudit (77%) compared with preintervention (33%) (p=0.046). Rescue medication was prescribed in 50% of postintervention and 65% of reaudit cases compared with 0% preintervention (p<0.001). Intermediate/long-acting insulin was appropriately amended more often in the postintervention versus preintervention period (75% vs 45%, p=0.041). Overall, VRIII was appropriate for the situation in 85% of cases.ConclusionsThe quality of perioperative VRIII prescribing practices improved following the proposed interventions, with prescribers more frequently using recommended safety measures such as ‘refer to paper chart’ and rescue medication. There was a marked sustained improvement in prescriber-initiated adjustment of oral diabetes medications and insulins. VRIII is occasionally administered unnecessarily in a subset of patients with type 2 diabetes and may be an area for further study.

We describe the development of a mini-library of human DNA fragments for use in an enquiry-based learning (EBL) undergraduate practical incorporating 'wet-lab' and bioinformatics tasks. In spite of the widespread emergence of the polymerase chain reaction (PCR), the cloning and analysis of DNA fragments in Escherichia coli remains a fundamental skill commonly taught to undergraduate biology students. The individual procedures are simple enough, but enthusiastic engagement of the students in practical exercises requires that a better learning experience be offered to them than the simple opportunity to participate. To address this issue we have prepared a mini-library of well-characterised fragments of human DNA in a small plasmid vector. We have generated high-quality sequence data for each end of the human DNAs and there are sufficient clones for around 80 students to each engage in a series of enquiry-based activities producing results that are unique to each student. This resource allows the students to experience the real-life integration of 'wet-lab' and bioinformatics approaches in modern molecular genetics. The experimental procedures are aimed at molecular genetics students commencing their second year of undergraduate study, but the materials could also be used with more advanced groups, up to and including masters degree level, by appropriate adaptation and extension of the range of experimental techniques used.