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The discovery of the genetic cause of an inflammatory disorder shows that, in winnowing down candidate variants obtained by DNA sequencing screens, geneticists have been (so to speak) throwing the baby out with the bath water.

BackgroundDifferent diagnostic and classification criteria are available for hereditary recurrent fevers (HRF)—familial Mediterranean fever (FMF), tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS), mevalonate kinase deficiency (MKD) and cryopyrin-associated periodic syndromes (CAPS)—and for the non-hereditary, periodic fever, aphthosis, pharyngitis and adenitis (PFAPA). We aimed to develop and validate new evidence-based classification criteria for HRF/PFAPA.MethodsStep 1: selection of clinical, laboratory and genetic candidate variables; step 2: classification of 360 random patients from the Eurofever Registry by a panel of 25 clinicians and 8 geneticists blinded to patients’ diagnosis (consensus ≥80%); step 3: statistical analysis for the selection of the best candidate classification criteria; step 4: nominal group technique consensus conference with 33 panellists for the discussion and selection of the final classification criteria; step 5: cross-sectional validation of the novel criteria.ResultsThe panellists achieved consensus to classify 281 of 360 (78%) patients (32 CAPS, 36 FMF, 56 MKD, 37 PFAPA, 39 TRAPS, 81 undefined recurrent fever). Consensus was reached for two sets of criteria for each HRF, one including genetic and clinical variables, the other with clinical variables only, plus new criteria for PFAPA. The four HRF criteria demonstrated sensitivity of 0.94–1 and specificity of 0.95–1; for PFAPA, criteria sensitivity and specificity were 0.97 and 0.93, respectively. Validation of these criteria in an independent data set of 1018 patients shows a high accuracy (from 0.81 to 0.98).ConclusionEurofever proposes a novel set of validated classification criteria for HRF and PFAPA with high sensitivity and specificity.

Cryopyrin-associated periodic syndrome (CAPS) is caused by mutations. Recently, somatic mosaicism has been reported in some CAPS patients who were previously classified as \"mutation-negative.\" We describe here the clinical and laboratory findings in eight British adult patients who presented with symptoms typical of CAPS other than an onset in mid-late adulthood. All patients underwent comprehensive clinical and laboratory investigations, including analysis of the gene using Sanger and amplicon-based deep sequencing (ADS) along with measurements of extracellular apoptosis-associated speck-like protein with CARD domain (ASC) aggregates. The clinical phenotype in all subjects was consistent with mid-spectrum CAPS, except a median age at disease onset of 50 years. Sanger sequencing of was non-diagnostic but ADS detected a somatic mutation in each case. In one patient, DNA isolated from blood demonstrated an increase in the mutant allele from 5 to 45% over 12 years. ASC aggregates in patients' serum measured during active disease were significantly higher than healthy controls. This series represents 8% of CAPS patients diagnosed in a single center, suggesting that acquired mutations may not be an uncommon cause of the syndrome and should be sought in all patients with late-onset symptoms otherwise compatible with CAPS. Steadily worsening CAPS symptoms in one patient were associated with clonal expansion of the mutant allele predominantly affecting myeloid cells. Two patients developed AA amyloidosis, which previously has only been reported in CAPS in association with life-long germline mutations.

BackgroundHereditary recurrent fevers (HRFs) are rare inflammatory diseases sharing similar clinical symptoms and effectively treated with anti-inflammatory biological drugs. Accurate diagnosis of HRF relies heavily on genetic testing.ObjectivesThis study aimed to obtain an experts’ consensus on the clinical significance of gene variants in four well-known HRF genes: MEFV, TNFRSF1A, NLRP3 and MVK.MethodsWe configured a MOLGENIS web platform to share and analyse pathogenicity classifications of the variants and to manage a consensus-based classification process. Four experts in HRF genetics submitted independent classifications of 858 variants. Classifications were driven to consensus by recruiting four more expert opinions and by targeting discordant classifications in five iterative rounds.ResultsConsensus classification was reached for 804/858 variants (94%). None of the unsolved variants (6%) remained with opposite classifications (eg, pathogenic vs benign). New mutational hotspots were found in all genes. We noted a lower pathogenic variant load and a higher fraction of variants with unknown or unsolved clinical significance in the MEFV gene.ConclusionApplying a consensus-driven process on the pathogenicity assessment of experts yielded rapid classification of almost all variants of four HRF genes. The high-throughput database will profoundly assist clinicians and geneticists in the diagnosis of HRFs. The configured MOLGENIS platform and consensus evolution protocol are usable for assembly of other variant pathogenicity databases. The MOLGENIS software is available for reuse at http://github.com/molgenis/molgenis; the specific HRF configuration is available at http://molgenis.org/said/. The HRF pathogenicity classifications will be published on the INFEVERS database at https://fmf.igh.cnrs.fr/ISSAID/infevers/.

Abstract Background Monogenic autoinflammatory diseases are caused by pathogenic variants in genes that regulate innate immune responses, and are characterized by sterile systemic inflammatory episodes. Since symptoms can overlap within this rapidly expanding disease category, accurate genetic diagnosis is of the utmost importance to initiate early inflammation-targeted treatment and prevent clinically significant or life-threatening complications. Initial recommendations for the genetic diagnosis of autoinflammatory diseases were limited to a gene-by-gene diagnosis strategy based on the Sanger method, and restricted to the 4 prototypic recurrent fevers (MEFV, MVK, TNFRSF1A, and NLRP3 genes). The development of best practices guidelines integrating critical recent discoveries has become essential. Methods The preparatory steps included 2 online surveys and pathogenicity annotation of newly recommended genes. The current guidelines were drafted by European Molecular Genetics Quality Network members, then discussed by a panel of experts of the International Society for Systemic Autoinflammatory Diseases during a consensus meeting. Results In these guidelines, we combine the diagnostic strength of next-generation sequencing and recommendations to 4 more recently identified genes (ADA2, NOD2, PSTPIP1, and TNFAIP3), nonclassical pathogenic genetic alterations, and atypical phenotypes. We present a referral-based decision tree for test scope and method (Sanger versus next-generation sequencing) and recommend on complementary explorations for mosaicism, copy-number variants, and gene dose. A genotype table based on the 5-category variant pathogenicity classification provides the clinical significance of prototypic genotypes per gene and disease. Conclusions These guidelines will orient and assist geneticists and health practitioners in providing up-to-date and appropriate diagnosis to their patients.

BackgroundIn minimally symptomatic early neuropathic disease where neurophysiology is normal, skin amyloid deposits have been demonstrated to be a marker of hereditary transthyretin (ATTRv) amyloid neuropathy with intraepidermal nerve fibre density (IENFD), a marker of disease progression.AimsTo study the utility of skin biopsy in the diagnosis of UK ATTRv patients and to assess its influence on accessing gene silencing therapy.Methods53 patients had skin biopsies performed between July 2021 and October 2022. These were stained for amyloid (typing by immunohistochemistry) and analysed for IENFD.ResultsA total of 59 skin biopsies were performed since July 2021. Neurophysiology was normal in 62% of cases. From available results, 48% of cases had an abnormal IENFD result, 37% had positive amyloid staining and 24% had both. These results allowed 27% of these patients to start gene silencing therapy.ConclusionSkin biopsy is a useful method for detecting amyloid deposits and small nerve fibre loss. Crucially, It allowed a substantial number of patients to start gene silencing therapy who would otherwise not have been eligible. As T60A and V122I are the commonest TTR variants in the UK, with patients often presentingwith cardiomyopathy, earlydiagnosisofamyloid neuropathy is imperative for therapy decisions.

BackgroundHereditary transthyretin (ATTRv) amyloidosis is known to be associated with spinal stenosis through deposition of amyloid in the ligamentum flavum (LF).AimsTo analyse lumbosacral spinal MRI changes and associated symptoms/signs in patients with ATTRv amyloidosis some of whom were subsequently treated with gene silencing therapy.MethodsWe performed a retrospective analysis of 48 ATTRv patients who had a lumbosacral MRI scan between 2001 and 2022.ResultsLF hypertrophy was demonstrated in 24 patients and 11 had symptoms of spinal claudication at baseline imaging. LF hypertrophy was present across multiple pathogenic TTR variants with the highest proportion in the T60A and V122I variants. 18 patients had LF hypertrophy with associated spinal stenosis, 5 of which were severe. 12 patients had repeat imaging. One patient was on gene silencing therapy for the full duration between scans and remained radiologically stable, while 6 patients who were not on disease-modifying therapy for the full duration between scans developed new evidence of LF hypertro- phy and/or worsening spinal changes.ConclusionLF hypertrophy is seen in association with multiple amyloidogenic TTR variants and can be associated with symptomatic spinal stenosis. Further studies are needed to assess if gene silencing therapy reduces LF hypertrophy and symptomatic spinal stenosis.

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. 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. 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%). 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.

ObjectivesWild-type transthyretin amyloid cardiomyopathy (wtATTR-CM) is a progressive and fatal condition. Although prognosis can be determined at the time of diagnosis according to National Amyloidosis Centre (NAC) transthyretin amyloidosis (ATTR) stage, the clinical course varies substantially between individuals. There are currently no established measures of rate of disease progression. Through systematic analysis of functional, biochemical and echocardiographic disease-related variables we aimed to identify prognostic markers of disease progression in wtATTR-CM.MethodsThis is a retrospective observational study of 432 patients with wtATTR-CM diagnosed at the UK NAC, none of whom received disease-modifying therapy. The association between mortality from the 12-month timepoint and change from diagnosis to 12 months in a variety of disease-related variables was explored using Cox regression.ResultsChange in N-terminal pro-B-type natriuretic peptide concentration (∆ NT-proBNP) at 12 months from diagnosis was the strongest predictor of ongoing mortality and was independent of both change in other disease-related variables (HR 1.04 per 500 ng/L increase (95% CI 1.01 to 1.07); p=0.003) and a range of known prognostic variables at the time of diagnosis (HR 1.07 per 500 ng/L increase (95% CI 1.02 to 1.13); p=0.007). An increase in NT-proBNP of >500 ng/L, >1000 ng/L and >2000 ng/L during the first year of follow-up occurred in 45%, 35% and 16% of patients, respectively.ConclusionChange in NT-proBNP concentration during the first year of follow-up is a powerful independent predictor of mortality in wtATTR-CM.

Monogenic autoinflammatory disorders (AIDs) are rare diseases caused by variants in genes regulating the innate immune system. The identification of the first four genes responsible for the prototype group of hereditary recurrent fevers prompted the development of genetic diagnosis, followed by external quality assessment and guidelines for the interpretation of sequence variants in these diseases. Recent changes in the diagnosis of genetic diseases, namely the implementation of next-generation sequencing (NGS), lead to discovery of the new genes associated with at least 40 novel AIDs, which revolutionized patient care and prognosis. However, these rapid advances resulted in nonstandardized molecular strategies that can influence genetic diagnosis and reporting of results. In order to assess factors, which may have an impact on performance and quality of results in the NGS era, we carried out an online survey among member laboratories of the European Molecular Genetics Quality Network, which highlighted different strategies being used and identified pitfalls that deserve discussion and improvement.