Social determinants of health inequalities in early phase clinical trials in Northern England.

BACKGROUND: Early phase clinical trials in Oncology represent a subspecialised area where UK patient selection is influenced by access to Experimental Cancer Medicine Centres (ECMCs). Equity of access with respect to social determinants of health (SDoH) were explored for two major ECMCs. METHODS: A retrospective cohort study including all referrals to Newcastle and Manchester ECMCs in 2021 was completed. Consent to screening or pre-screening was stratified against SDoH characteristics, including: Index of Multiple Deprivation (IMD) decile, ethnicity and distance to centre. RESULTS: 1243 patients were referred for trials. IMD quintile 1 (most deprived) patients had reduced likelihood of referral compared to expected population models (OR, 0.67; 95% CI: 0.55 to 0.80, p = <0.0001). IMD quintile 5 (least deprived) had increased likelihood of referral (OR, 1.46; 95% CI: 1.17 to 1.82, p = 0.0007). Living beyond median distance from Manchester reduced the likelihood of consenting to trials (OR, 0.72; 95% CI: 0.55 to 0.94, p = 0.015). Ethnicity data represented a White British propensity. CONCLUSIONS: Inequalities in socioeconomic and geographic factors influence referral and enrolment to early phase clinical trials in Northern England. This has implications for equity of access and generalisability of trial results internationally and warrants further study.

Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL): Report of a new family and a novel DARS2 mutation.

Background: LBSL is a mitochondrial disorder caused by mutations in the mitochondrial aspartyl-tRNA synthetase gene DARS2, resulting in a distinctive pattern on brain magnetic resonance imaging (MRI) and spectroscopy. Clinical presentation varies from severe infantile to chronic, slowly progressive neuronal deterioration in adolescents or adults. Most individuals with LBSL are compound heterozygous for one splicing defect in an intron 2 mutational hotspot and a second defect that could be a missense, non-sense, or splice site mutation or deletion resulting in decreased expression of the full-length protein. Aim: To present a new family with two affected members with LBSL and report a novel DARS2 mutation. Results: An 8-year-old boy (Patient 1) was referred due to headaches and abnormal MRI, suggestive of LBSL. Genetic testing revealed a previously reported c.492 + 2 T > C mutation in the DARS2 gene. Sanger sequencing uncovered a novel variant c.228-17C > G in the intron 2 hotspot. Family studies found the same genetic changes in an asymptomatic 4-year-old younger brother (Patient 2), who was found on follow-up to have an abnormal MRI. mRNA extracted from patients' fibroblasts showed that the c.228-17C > G mutation caused skipping of exon 3 resulting in lower DARS2 mRNA level. Complete absence of DARS2 protein was also found in both patients. Summary: We present a new family with two children affected with LBSL and describe a novel mutation in the DARS2 intron 2 hotspot. Despite findings of extensive white matter disease in the brain and spine, the proband in this family presented only with headaches, while the younger sibling, who also had extensive white matter changes, was asymptomatic. Our in-vitro results confirmed skipping of exon 3 in patients and family members carrying the intron 2 variant, which is consistent with previous reported mutations in intron 2 hotspots. DARS2 mRNA and protein levels were also reduced in both patients, further supporting the pathogenicity of the novel variant.

ECHS1 disease in two unrelated families of Samoan descent: Common variant - rare disorder.

Mutations in the short-chain enoyl-CoA hydratase (SCEH) gene, ECHS1, cause a rare autosomal recessive disorder of valine catabolism. Patients usually present with developmental delay, regression, dystonia, feeding difficulties, and abnormal MRI with bilateral basal ganglia involvement. We present clinical, biochemical, molecular, and functional data for four affected patients from two unrelated families of Samoan descent with identical novel compound heterozygous mutations. Family 1 has three affected boys while Family 2 has an affected daughter, all with clinical and MRI findings of Leigh syndrome and intermittent episodes of acidosis and ketosis. WES identified a single heterozygous variant in ECHS1 at position c.832G > A (p.Ala278Thr). However, western blot revealed significantly reduced ECHS1 protein for all affected family members. Decreased SCEH activity in fibroblasts and a mild increase in marker metabolites in urine further supported ECHS1 as the underlying gene defect. Additional investigations at the DNA (aCGH, WGS) and RNA (qPCR, RT-PCR, RNA-Seq, RNA-Array) level identified a silent, common variant at position c.489G > A (p.Pro163=) as the second mutation. This substitution, present at high frequency in the Samoan population, is associated with decreased levels of normally spliced mRNA. To our understanding, this is the first report of a novel, hypomorphic allele c.489G > A (p.Pro163=), associated with SCEH deficiency.

Comparing febrile children presenting on and off antibiotics to the emergency department: a retrospective cohort study.

BACKGROUND: It is not yet known how antibiotics may affect Serious Bacterial Infections (SBI). Our aim is to describe the presentation, management, and serious bacterial infections (SBI) of febrile children on or off antibiotics. METHODS: Retrospective, cohort study of febrile Emergency Department patients, 0-36 months of age, at a single institution, between 2009and 2012. RESULTS: Seven hundred fifty-three patients were included: 584 in the No-Antibiotics group and 169 (22%) in the Antibiotics group. Age and abnormal lung sounds were predictors for being on antibiotics (OR 2.00 [95% CI 1.23-3.25] and OR 1.04 [95% CI 1.02-1.06] respectively) while female gender, and lower temperatures were negative predictors (OR 0.68 [95%0.47-0.98] and OR 0.47 [95% CI 0.32-0.67] respectively). Antibiotics were prescribed by a physician 89% of the time; the most common one being Amoxicillin/Clavulanic Acid (39%). The antibiotic group got more blood tests (57% vs 45%) and Chest X-Rays (37% vs 25%). Overall, the percent of SBIs (and pneumonias) was statistically the same in both groups (6.5% in the No-antibiotic group VS 3.6%). CONCLUSIONS: Children presenting on antibiotics and off antibiotics were significantly different in their presentation and management, although the overall percentages of SBI were similar in each group. Further investigations into this subgroup of febrile children are needed.

Thrombus stability explains the factor V Leiden paradox: a mouse model.

Humans carrying the factor V Leiden (FVL) variant have a fivefold increased risk for venous thrombosis. However, incidence of deep vein thrombosis (DVT) is proportionally greater than that of pulmonary embolism (PE) in these individuals. This is known as the FVL paradox. We hypothesized that the rate of initial DVT development is similar in FVL and noncarriers, but thrombi in FVL carriers are more stable and develop into a clinically significant DVT more often than in noncarriers. To test this, we induced thrombi in the femoral vein of wild-type (WT), heterozygous (F5L/+), and FVL homozygous (F5L/L) mice. Using intravital microscopy, thrombus size and embolization were visualized and emboli in the lungs were quantified. Compared with WT, femoral vein thrombi in F5L/+ and F5L/L mice were larger and embolized less. Total and large embolic events, the percentage of thrombus that embolized, and PE burden were significantly decreased in F5L/L mice. This suggests that in noncarriers (reflected by WT), a minor injury initially resulting in a small DVT tends to remain small and asymptomatic because of the embolization of the otherwise growing thrombus. Alternatively, the same insult in people with FVL (reflected by F5L/L) leads to thrombus growth as a result of less embolization, and thus symptomatic DVT development.

Novel mutations in the mitochondrial complex I assembly gene NDUFAF5 reveal heterogeneous phenotypes.

Primary mitochondrial complex I deficiency is the most common defect of the mitochondrial respiratory chain. It is caused by defects in structural components and assembly factors of this large protein complex. Mutations in the assembly factor NDUFAF5 are rare, with only five families reported to date. This study provides clinical, biochemical, molecular and functional data for four unrelated additional families, and three novel pathogenic variants. Three cases presented in infancy with lactic acidosis and classic Leigh syndrome. One patient, however, has a milder phenotype, with symptoms starting at 27 months and a protracted clinical course with improvement and relapsing episodes. She is homozygous for a previously reported mutation, p.Met279Arg and alive at 19 years with mild neurological involvement, normal lactate but abnormal urine organic acids. We found the same mutation in one of our severely affected patients in compound heterozygosity with a novel p.Lys52Thr mutation. Both patients with p.Met279Arg are of Taiwanese descent and had severe hyponatremia. Our third and fourth patients, both Caucasian, shared a common, newly described, missense mutation p.Lys109Asn which we show induces skipping of exon 3. Both Caucasian patients were compound heterozygotes, one with a previously reported Ashkenazi founder mutation while the other was negative for additional exonic variants. Whole genome sequencing followed by RNA studies revealed a novel deep intronic variant at position c.223-907A>C inducing an exonic splice enhancer. Our report adds significant new information to the mutational spectrum of NDUFAF5, further delineating the phenotypic heterogeneity of this mitochondrial defect.

Comparison of the effect of dabigatran and dalteparin on thrombus stability in a murine model of venous thromboembolism.

UNLABELLED: ESSENTIALS: Does thrombus stability alter the presentation of venous thromboembolism and do anticoagulants alter this? In a murine model, we imaged a femoral vein thrombus and quantified emboli in the pulmonary arteries. Dabigatran decreases thrombus stability via factor XIII increasing embolization and pulmonary emboli. This cautions against the unapproved use of dabigatran for acute initial treatment of deep vein thrombosis. BACKGROUND: Venous thromboembolism (VTE) is a collective term for deep vein thrombosis (DVT) and pulmonary embolism (PE). Thrombus instability possibly contributes to progression of DVT to PE, and direct thrombin inhibitors (DTIs) may alter this. AIM: To develop a model to assess thrombus stability and its link to PE burden, and identify whether DTIs, in contrast to low-molecular-weight heparin (LMWH), alter this correlation. METHODS: Twelve minutes after ferric chloride-induced thrombus formation in the femoral vein of female mice, saline, dalteparin (LMWH) or dabigatran (DTI) was administered. Thrombus size and embolic events breaking off from the thrombus were quantified before treatment and at 10-min intervals after treatment for 2 h using intravital videomicroscopy. Lungs were stained for the presence of PE. RESULTS: Thrombus size was similar over time and between treatment groups. Total and large embolic events and pulmonary emboli were highest after treatment with dabigatran. Variations in amounts of pulmonary embolic events were not attributed to variations in thrombus size. Large embolic events correlated with the number of emboli per lung slice independent of treatment. Embolization in factor XIII deficient (FXIII(-/-) ) saline-treated mice was greater than that in wild-type (WT) saline-treated mice, but was similar to WT dabigatran-treated mice. CONCLUSION: We have developed a mouse model of VTE that can quantify emboli and correlate this with PE burden. Consistent with clinical data, dabigatran, a DTI, acutely decreases thrombus stability and increases PE burden compared with LMWH or saline, which is a FXIII-dependent effect.

Aberrant neutrophil trafficking and metabolic oscillations in severe pyoderma gangrenosum.

Having previously associated metabolic oscillations with cell locomotion, we hypothesized that patients with abnormalities in neutrophil trafficking may display aberrant intracellular oscillations. A pyoderma gangrenosum patient exhibiting aberrant leukocyte trafficking in vivo and skin ulceration without infection was identified. This patient's neutrophils constitutively overexpressed and clustered the leukocyte integrins CR3 and CR4 and failed to display appropriate integrin-to-GPI receptor interactions. Increased levels of tyrosine phosphorylation were observed. NAD(P)H oscillations, which are sinusoidal in normals, were chaotic with multiple frequency components in this patient's neutrophils. Normal cell shape and sinusoidal NAD(P)H oscillations were restored by providing a pulsed electric field to drive metabolic oscillations and by temperature reduction. N-acetyl-D-glucosamine disrupted CR3 clusters and sinusoidal NAD(P)H oscillations returned. Anecdotal reports suggest that local hypothermia is clinically useful for this patient. These data define the first metabolic oscillation-associated disease and suggest that pyoderma gangrenosum can be classified as a dynamical disease at the cellular level.

Aberrant integrin (CR4; alpha(x)beta2; CD11c/CD18) oscillations on neutrophils in a mild form of pyoderma gangrenosum.

We have previously shown that the beta2 integrins CR3 and CR4 physically and functionally interact with urokinase receptors (uPAR) on neutrophil plasma membranes in an oscillatory fashion. In this study we have analyzed neutrophils from patient SC, a 34 y old African American female, with aberrant skin window results and recurrent perianal abscesses and pretibial lesions diagnosed as pyoderma gangrenosum. Although untreated migrating normal neutrophils exhibited 20 s sinusoidal oscillations in CR4-uPAR proximity, neutrophils from SC demonstrated a faster oscillation (10 s) in the form of a flyback sawtooth wave. This waveform mimicked that observed for normal neutrophils treated with subsaturating doses of the kinase inhibitors staurosporine, genistein, and erbstatin. As beta2 integrins are regulated by phosphorylation, we tested the hypothesis that the aberrant CR4-uPAR proximity oscillations seen in SC's neutrophils are due to defective kinase activity that might be balanced by a decrease in phosphatase activity. When SC's cells are exposed to subsaturating concentrations of the phosphatase inhibitor pervanadate, this caused the CR4-uPAR oscillations to become sinusoidal in shape with a 20 s period, as seen in normal migrating neutrophils. Although SC's neutrophils were deficient in spontaneous and N-formyl-methionyl-leucyl-phenylalanine-induced polarization, 0.5 microM pervanadate returned cell polarization to nearly normal levels, thus paralleling the acquisition of normal receptor interactions. Inasmuch as SC's cellular phenotype is mimicked by kinase inhibitors and corrected by phosphatase inhibitors, we suggest that a mutation(s) affecting the kinetics of intracellular signaling enzymes, but not blocking the pathway per se, may be responsible for this clinical state.