Paediatric Behçet's disease: a UK tertiary centre experience

There are currently limited data regarding paediatric Behçet's disease (BD), particularly in the UK. We describe the clinical spectrum, treatment and outcome of BD, and explore the relative sensitivities of the criteria for the diagnosis of BD in a UK paediatric cohort. Single retrospective case note review of children with a clinical diagnosis of BD presenting between 1987 and 2012. Demographics, clinical features, treatment and outcomes were recorded. The sensitivities of the International Study Group (ISG) and International Criteria for BD (ICBD) criteria were explored. BD disease activity was calculated using the Behçet's Disease Activity Index (BDAI). Forty-six patients (22 male) were identified. Median age of onset was 4.87 (0.04-15.71) years; median time to diagnosis was 3.74 (0.25-13.48) years. Clinical features were recurrent oral ulceration (97.8 %), recurrent genital ulceration (73.9 %), gastrointestinal (58.7 %), musculoskeletal (47.83 %), cutaneous (23.9 %) involvement and uveitis (2 %). Recurrent genital ulceration was more common in female patients (P = 0.044). Thirty-seven patients (80.4 %) fulfilled the ICBD criteria; only 12 patients (26.1 %) fulfilled the ISG criteria. BDAI score at diagnosis was 7/20 (0-10/20) and significantly decreased to 5/20 (0-9/20) (P < 0.0001) at latest follow-up. The commonest systemic treatment was colchicine (76.1 %); anti-TNFα treatment was reserved for severe cases (15.5 %). Paediatric BD in the UK may present very early in life, sometimes with a family history, and with a low incidence of ocular involvement. Diagnostic delay is common. The majority of our patients required systemic therapy; anti-TNFα was reserved for severe cases and has largely superseded the use of thalidomide

Early-Onset Juvenile SLE Associated With a Novel Mutation in Protein Kinase C δ

Juvenile systemic lupus erythematosus (jSLE) is rare before 5 years of age. Monogenic causes are suspected in cases of very early onset jSLE particularly in the context of a family history and/or consanguinity. We performed whole-exome sequencing and homozygosity mapping in the siblings presented with early-onset jSLE. A novel homozygous missense mutation in protein kinase C delta (c.1294G>T; p.Gly432Trp) was identified in both patients. One patient showed a marked clinical response and resolution inflammation with rituximab therapy. This report demonstrates the clinical importance of identifying monogenic causes of rare disease to provide a definitive diagnosis, help rationalize treatment, and facilitate genetic counseling

Adenosine Deaminase Two and Immunoglobulin M Accurately Differentiate Adult Sneddon's Syndrome of Unknown Cause

BACKGROUND: The association that exists between livedo reticularis (LR) and stroke is known as Sneddon's syndrome (SnS). The disorder is classified as primary SnS (PSnS), if the cause remains unknown and secondary SnS. The condition is rare and it occurs mainly sporadically. In 2014, 2 independent teams described a new genetic disorder with childhood-onset, which was called deficiency of adenosine deaminase 2 (DADA2), characterized by recurrent fevers and vascular pathologic features that included LR and stroke. All the patients carried recessively inherited mutations in cat eye syndrome chromosome region candidate 1 gene (CECR1), encoding the adenosine deaminase 2 (ADA2) protein. Genetic testing is the standard for the diagnosis of DADA2. However, the diagnostic accuracy of more affordable laboratorial analysis in CECR1-mutated individuals remains to be established. We aim to determine whether plasma ADA2 activity and serum immunoglobulin M (IgM) levels can distinguish (1) DADA2 from other adult patients within the SnS spectrum, and (2) healthy CECR1 heterozygous (HHZ) from healthy controls (HC). METHODS: ADA2 activity in plasma and serum IgM concentrations was measured in adult patients within the SnS spectrum, healthy first-degree relatives and HC. Genetic results were used as the reference standard. The primary outcome measures were sensitivity and specificity derived from receiver operating curve analysis. RESULTS: A total of 73 participants were included in the study: 26 patients with PSnS with no CECR1 mutation (PSnS), 6 bi-allelic (DADA2 patients) and 7 HHZ CECR1 mutations and 34 HC. Plasma ADA2 activity and serum IgM levels were significantly lower in DADA2 patients than in PSnS. With the use of the best indexes, plasma ADA2 activity differentiated PSnS from DADA2 with a sensitivity and specificity of 100.0% and HHZ from HC with a sensitivity of 97.1% and specificity of 85.7%. Serum IgM levels also differentiated PSnS from DADA2 with a sensitivity of 85.2% and specificity of 83.3%. CONCLUSION: Serum IgM levels might be used as a triage tool and plasma ADA2 activity performs perfectly as a diagnostic test for DADA2 in adult patients within the SnS spectrum. ADA2 activity in plasma also reliably distinguishes HHZ from HC.info:eu-repo/semantics/publishedVersio

Autoinflammatory periodic fever, immunodeficiency, and thrombocytopenia (PFIT) caused by mutation in actinregulatory gene WDR1

The importance of actin dynamics in the activation of the inflammasome is becoming increasingly apparent. IL-1β, which is activated by the inflammasome, is known to be central to the pathogenesis of many monogenic autoinflammatory diseases. However, evidence from an autoinflammatory murine model indicates that IL-18, the other cytokine triggered by inflammasome activity, is important in its own right. In this model, autoinflammation was caused by mutation in the actin regulatory gene WDR1 We report a homozygous missense mutation in WDR1 in two siblings causing periodic fevers with immunodeficiency and thrombocytopenia. We found impaired actin dynamics in patient immune cells. Patients had high serum levels of IL-18, without a corresponding increase in IL-18-binding protein or IL-1β, and their cells also secreted more IL-18 but not IL-1β in culture. We found increased caspase-1 cleavage within patient monocytes indicative of increased inflammasome activity. We transfected HEK293T cells with pyrin and wild-type and mutated WDR1 Mutant protein formed aggregates that appeared to accumulate pyrin; this could potentially precipitate inflammasome assembly. We have extended the findings from the mouse model to highlight the importance of WDR1 and actin regulation in the activation of the inflammasome, and in human autoinflammation

Clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis.

BACKGROUND: Monogenic autoinflammatory diseases (AID) are a rapidly expanding group of genetically diverse but phenotypically overlapping systemic inflammatory disorders associated with dysregulated innate immunity. They cause significant morbidity, mortality and economic burden. Here, we aimed to develop and evaluate the clinical impact of a NGS targeted gene panel, the "Vasculitis and Inflammation Panel" (VIP) for AID and vasculitis. METHODS: The Agilent SureDesign tool was used to design 2 versions of VIP; VIP1 targeting 113 genes, and a later version, VIP2, targeting 166 genes. Captured and indexed libraries (QXT Target Enrichment System) prepared for 72 patients were sequenced as a multiplex of 16 samples on an Illumina MiSeq sequencer in 150bp paired-end mode. The cohort comprised 22 positive control DNA samples from patients with previously validated mutations in a variety of the genes; and 50 prospective samples from patients with suspected AID in whom previous Sanger based genetic screening had been non-diagnostic. RESULTS: VIP was sensitive and specific at detecting all the different types of known mutations in 22 positive controls, including gene deletion, small INDELS, and somatic mosaicism with allele fraction as low as 3%. Six/50 patients (12%) with unclassified AID had at least one class 5 (clearly pathogenic) variant; and 11/50 (22%) had at least one likely pathogenic variant (class 4). Overall, testing with VIP resulted in a firm or strongly suspected molecular diagnosis in 16/50 patients (32%). CONCLUSIONS: The high diagnostic yield and accuracy of this comprehensive targeted gene panel validate the use of broad NGS-based testing for patients with suspected AID

Dolutegravir twice-daily dosing in children with HIV-associated tuberculosis: a pharmacokinetic and safety study within the open-label, multicentre, randomised, non-inferiority ODYSSEY trial

Background: Children with HIV-associated tuberculosis (TB) have few antiretroviral therapy (ART) options. We aimed to evaluate the safety and pharmacokinetics of dolutegravir twice-daily dosing in children receiving rifampicin for HIV-associated TB. Methods: We nested a two-period, fixed-order pharmacokinetic substudy within the open-label, multicentre, randomised, controlled, non-inferiority ODYSSEY trial at research centres in South Africa, Uganda, and Zimbabwe. Children (aged 4 weeks to <18 years) with HIV-associated TB who were receiving rifampicin and twice-daily dolutegravir were eligible for inclusion. We did a 12-h pharmacokinetic profile on rifampicin and twice-daily dolutegravir and a 24-h profile on once-daily dolutegravir. Geometric mean ratios for trough plasma concentration (Ctrough), area under the plasma concentration time curve from 0 h to 24 h after dosing (AUC0–24 h), and maximum plasma concentration (Cmax) were used to compare dolutegravir concentrations between substudy days. We assessed rifampicin Cmax on the first substudy day. All children within ODYSSEY with HIV-associated TB who received rifampicin and twice-daily dolutegravir were included in the safety analysis. We described adverse events reported from starting twice-daily dolutegravir to 30 days after returning to once-daily dolutegravir. This trial is registered with ClinicalTrials.gov (NCT02259127), EudraCT (2014–002632-14), and the ISRCTN registry (ISRCTN91737921). Findings: Between Sept 20, 2016, and June 28, 2021, 37 children with HIV-associated TB (median age 11·9 years [range 0·4–17·6], 19 [51%] were female and 18 [49%] were male, 36 [97%] in Africa and one [3%] in Thailand) received rifampicin with twice-daily dolutegravir and were included in the safety analysis. 20 (54%) of 37 children enrolled in the pharmacokinetic substudy, 14 of whom contributed at least one evaluable pharmacokinetic curve for dolutegravir, including 12 who had within-participant comparisons. Geometric mean ratios for rifampicin and twice-daily dolutegravir versus once-daily dolutegravir were 1·51 (90% CI 1·08–2·11) for Ctrough, 1·23 (0·99–1·53) for AUC0–24 h, and 0·94 (0·76–1·16) for Cmax. Individual dolutegravir Ctrough concentrations were higher than the 90% effective concentration (ie, 0·32 mg/L) in all children receiving rifampicin and twice-daily dolutegravir. Of 18 children with evaluable rifampicin concentrations, 15 (83%) had a Cmax of less than the optimal target concentration of 8 mg/L. Rifampicin geometric mean Cmax was 5·1 mg/L (coefficient of variation 71%). During a median follow-up of 31 weeks (IQR 30–40), 15 grade 3 or higher adverse events occurred among 11 (30%) of 37 children, ten serious adverse events occurred among eight (22%) children, including two deaths (one tuberculosis-related death, one death due to traumatic injury); no adverse events, including deaths, were considered related to dolutegravir. Interpretation: Twice-daily dolutegravir was shown to be safe and sufficient to overcome the rifampicin enzyme-inducing effect in children, and could provide a practical ART option for children with HIV-associated TB

Neuropsychiatric manifestations and sleep disturbances with dolutegravir-based antiretroviral therapy versus standard of care in children and adolescents: a secondary analysis of the ODYSSEY trial

BACKGROUND: Cohort studies in adults with HIV showed that dolutegravir was associated with neuropsychiatric adverse events and sleep problems, yet data are scarce in children and adolescents. We aimed to evaluate neuropsychiatric manifestations in children and adolescents treated with dolutegravir-based treatment versus alternative antiretroviral therapy. METHODS: This is a secondary analysis of ODYSSEY, an open-label, multicentre, randomised, non-inferiority trial, in which adolescents and children initiating first-line or second-line antiretroviral therapy were randomly assigned 1:1 to dolutegravir-based treatment or standard-of-care treatment. We assessed neuropsychiatric adverse events (reported by clinicians) and responses to the mood and sleep questionnaires (reported by the participant or their carer) in both groups. We compared the proportions of patients with neuropsychiatric adverse events (neurological, psychiatric, and total), time to first neuropsychiatric adverse event, and participant-reported responses to questionnaires capturing issues with mood, suicidal thoughts, and sleep problems. FINDINGS: Between Sept 20, 2016, and June 22, 2018, 707 participants were enrolled, of whom 345 (49%) were female and 362 (51%) were male, and 623 (88%) were Black-African. Of 707 participants, 350 (50%) were randomly assigned to dolutegravir-based antiretroviral therapy and 357 (50%) to non-dolutegravir-based standard-of-care. 311 (44%) of 707 participants started first-line antiretroviral therapy (ODYSSEY-A; 145 [92%] of 157 participants had efavirenz-based therapy in the standard-of-care group), and 396 (56%) of 707 started second-line therapy (ODYSSEY-B; 195 [98%] of 200 had protease inhibitor-based therapy in the standard-of-care group). During follow-up (median 142 weeks, IQR 124–159), 23 participants had 31 neuropsychiatric adverse events (15 in the dolutegravir group and eight in the standard-of-care group; difference in proportion of participants with ≥1 event p=0·13). 11 participants had one or more neurological events (six and five; p=0·74) and 14 participants had one or more psychiatric events (ten and four; p=0·097). Among 14 participants with psychiatric events, eight participants in the dolutegravir group and four in standard-of-care group had suicidal ideation or behaviour. More participants in the dolutegravir group than the standard-of-care group reported symptoms of self-harm (eight vs one; p=0·025), life not worth living (17 vs five; p=0·0091), or suicidal thoughts (13 vs none; p=0·0006) at one or more follow-up visits. Most reports were transient. There were no differences by treatment group in low mood or feeling sad, problems concentrating, feeling worried or feeling angry or aggressive, sleep problems, or sleep quality. INTERPRETATION: The numbers of neuropsychiatric adverse events and reported neuropsychiatric symptoms were low. However, numerically more participants had psychiatric events and reported suicidality ideation in the dolutegravir group than the standard-of-care group. These differences should be interpreted with caution in an open-label trial. Clinicians and policy makers should consider including suicidality screening of children or adolescents receiving dolutegravir

Monogenic vasculitis of the young

Vasculitis is characterized by inflammation of the blood vessel wall, which can lead to vascular occlusion, ischaemia, and end-organ injury. The exact causes of most of the primary systemic vasculitides (PSV) are not fully understood; a monogenic cause is strongly suspected in a minority of cases, particularly when similar features occur in first-degree relatives; and/or disease onset is very early in life. The central hypothesis to this thesis is that there are novel monogenic causes of vasculitis affecting the young that may be revealed using next generation sequencing (NGS). The aim of this study was therefore to: 1. use NGS to identify novel candidate genes that could cause vasculitis in children; and 2. to study the potential pathogenic role of any candidate genes identified. Seven probands from 6 families (Sielicka et al.) with different phenotypes of vasculitis were recruited. Whole exome sequencing (WES) was performed in all probands. Homozygous or compound heterozygous loss-of-function mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), which encodes adenosine deaminase type 2, were identified in 4 probands of family A, B and C causing deficiency of adenosine deaminase type 2 (DADA2). Clinical features of DADA2 resemble polyarteritis nodosa. Further investigation in relatives of the probands, and a cohort of childhood PAN revealed an additional 17 affected individuals, 8 of whom were asymptomatic at the time of study. The main clinical features of DADA2 were: cutaneous involvement (66.7%), neurological involvement (52.4%) and immunological involvement (33.3%). Disease was well controlled in 11 patients using anti-TNF-α therapy. Functional studies revealed that ADA2 enzyme activity and CECR1 mRNA expression were decreased in DADA2 in all subjects with homozygous or compound heterozygous mutations (irrespective of whether they were symptomatic or not); and revealed important age-dependent physiological changes in ADA2 activity of healthy controls. DADA2 affects the function and polarisation of macrophages, and may represent a novel vasoprotective pathway. A novel homozygous missense mutation in Protein Kinase C Delta gene (PRKCD) c.1294G>T;p.Gly432Trp was identified in the proband of family D and her sister, who presented with early onset juvenile systemic lupus erythematosus. PRKCD encodes protein kinase C delta, which plays a role in apoptosis and proliferation of multiple cells, including B lymphocytes. The proband of family E presented with early onset cutaneous leukocytoclastic vasculitis, and recurrent bacterial infections. Immunological studies revealed abnormal functional complement assay. A novel homozygous mutation in the complement factor I gene (CFI) was identified. Complement factor I is a C3b/C4b inactivator. The phenotype described herein is therefore novel, since cutaneous leukocytoclastic vasculitis is not typical of CFI deficiency, although recurrent bacterial infections are recognized. The proband of family F presented with digital vasculitis, recurrent infections, and absence of serum C1q. C1q is a protein in the classical complement pathway and mainly produced by macrophages. No mutation in C1q genes was identified using standard Sanger sequencing, or from WES. RNA sequencing also revealed no mutation in any C1q gene; and mRNA expression of all 3 C1q genes was normal. C1q staining of macrophages (using confocal microscopy and intracellular flow cytometric analysis) revealed intracellular accumulation of C1q, suggesting a new syndrome of impaired C1q release, and functional C1q deficiency. These studies confirm my central hypothesis that monogenic vasculitides are an important clinical concern in some children; and yet again demonstrate the diagnostic utility of NGS. Future studies leading directly from the work described in this thesis are also considered