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.
The small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We ...recently demonstrated the existence of a long-lived pool of cycling stem cells defined by
Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target
Achaete scute-like 2 (
Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the
Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.
A novel autoinflammatory syndrome was recently described in male patients who harbored somatic mutations in the X-chromosomal UBA1 gene. These patients were characterized by adult-onset, ...treatment-refractory inflammation with fever, cytopenia, dysplastic bone marrow, vacuoles in myeloid and erythroid progenitor cells, cutaneous and pulmonary inflammation, chondritis, and vasculitis, which is abbreviated as VEXAS.
This study aimed to (retrospectively) diagnose VEXAS in patients who had previously been registered as having unclassified autoinflammation. We furthermore aimed to describe clinical experiences with this multifaceted, complex disease.
A systematic reanalysis of whole-exome sequencing data from a cohort of undiagnosed patients with autoinflammation from academic hospitals in The Netherlands was performed. When no sequencing data were available, targeted Sanger sequencing was applied in cases with high clinical suspicion of VEXAS.
A total of 12 male patients who carried mutations in UBA1 were identified. These patients presented with adult-onset (mean age 67 years, range 47-79 years) autoinflammation with systemic symptoms, elevated inflammatory parameters, and multiorgan involvement, most typically involving the skin and bone marrow. Novel features of VEXAS included interstitial nephritis, cardiac involvement, stroke, and intestinal perforation related to treatment with tocilizumab. Although many types of treatment were initiated, most patients became treatment-refractory, with a high mortality rate of 50%.
VEXAS should be considered in the differential diagnosis of males with adult-onset autoinflammation characterized by systemic symptoms and multiorgan involvement. Early diagnosis can prevent unnecessary diagnostic procedures and provide better prognostic information and more suitable treatment options, including stem cell transplantation.
Abstract
Objectives
The number of innate immune system disorders classified as systemic autoinflammatory diseases (SAID) has increased in recent years. More than 70% of patients with clinical ...manifestations of SAID did not receive a molecular diagnosis, thus being classed as so-called undifferentiated or undefined SAID (uSAID). The aim of the present study was to evaluate a next-generation sequencing (NGS)-based clinically oriented protocol in patients with uSAID.
Methods
We designed a NGS panel that included 41 genes clustered in seven subpanels. Patients with uSAID were classified into different groups according to their clinical features and sequenced for the coding portions of the 41 genes.
Results
Fifty patients were enrolled in the study. Thirty-four patients (72%) displayed recurrent fevers not consistent with a PFAPA phenotype. Sixteen patients displayed a chronic inflammatory disease course. A total of 100 gene variants were found (mean 2 per patient; range 0–6), a quarter of which affected suspected genes. Mutations with a definitive diagnostic impact were detected in two patients. Patients with genetically negative recurrent fevers displayed a prevalent gastrointestinal, skin and articular involvement. Patients responded to steroids on demands (94%) and colchicine, with a response rate of 78%.
Conclusion
Even with a low molecular diagnostic rate, a NGS-based approach is able to provide a final diagnosis in a proportion of uSAID patients with evident cost-effectiveness. It also allows the identification of a subgroup of genetically negative patients with recurrent fever responding to steroid on demand and colchicine.
Autoinflammation is the standard mechanism seen in systemic autoinflammatory disease (SAID) patients. This study aimed to investigate the effect of a candidate miRNA, miR-30e-3p, which was identified ...in our previous study, on the autoinflammation phenotype seen in SAID patients and to analyze its expression in a larger group of European SAID patients. We examined the potential anti-inflammatory effect of miR-30e-3p, which we had defined as one of the differentially expressed miRNAs in microarray analysis involved in inflammation-related pathways. This study validated our previous microarray results of miR-30e-3p in a cohort involving European SAID patients. We performed cell culture transfection assays for miR-30e-3p. Then, in transfected cells, we analyzed expression levels of pro-inflammatory genes; IL-1β, TNF-α, TGF-β, and MEFV. We also performed functional experiments, caspase-1 activation by fluorometric assay kit, apoptosis assay by flow cytometry, and cell migration assays by wound healing and filter system to understand the possible effect of miR-30e-3p on inflammation. Following these functional assays, 3'UTR luciferase activity assay and western blotting were carried out to identify the target gene of the aforementioned miRNA. MiR-30e-3p was decreased in severe European SAID patients like the Turkish patients. The functional assays associated with inflammation suggested that miR-30e-3p has an anti-inflammatory effect. 3'UTR luciferase activity assay demonstrated that miR-30e-3p directly binds to interleukin-1-beta (IL-1β), one of the critical molecules of inflammatory pathways, and reduces both RNA and protein levels of IL-1β. miR-30e-3p, which has been associated with IL-1β, a principal component of inflammation, might be of potential diagnostic and therapeutic value for SAIDs.
Key Messages
miR-30e-3p, which targets IL-1β, could have a role in the pathogenesis of SAID patients.
miR-30e-3p has a role in regulating inflammatory pathways like migration, caspase-1 activation.
miR-30e-3p has the potential to be used for future diagnostic and therapeutic approaches.
The self-renewing epithelium of the small intestine is ordered into stem/progenitor crypt compartments and differentiated villus compartments. Recent evidence indicates that the Wnt cascade is the ...dominant force in controlling cell fate along the crypt-villus axis. Here we show a rapid, massive conversion of proliferative crypt cells into post-mitotic goblet cells after conditional removal of the common Notch pathway transcription factor CSL/RBP-J (ref. 2). We obtained a similar phenotype by blocking the Notch cascade with a γ-secretase inhibitor. The inhibitor also induced goblet cell differentiation in adenomas in mice carrying a mutation of the Apc tumour suppressor gene. Thus, maintenance of undifferentiated, proliferative cells in crypts and adenomas requires the concerted activation of the Notch and Wnt cascades. Our data indicate that γ-secretase inhibitors, developed for Alzheimer's disease, might be of therapeutic benefit in colorectal neoplastic disease.
Each year diagnostic laboratories in the Netherlands profile thousands of individuals for heritable disease using next‐generation sequencing (NGS). This requires pathogenicity classification of ...millions of DNA variants on the standard 5‐tier scale. To reduce time spent on data interpretation and increase data quality and reliability, the nine Dutch labs decided to publicly share their classifications. Variant classifications of nearly 100,000 unique variants were catalogued and compared in a centralized MOLGENIS database. Variants classified by more than one center were labeled as “consensus” when classifications agreed, and shared internationally with LOVD and ClinVar. When classifications opposed (LB/B vs. LP/P), they were labeled “conflicting”, while other nonconsensus observations were labeled “no consensus”. We assessed our classifications using the InterVar software to compare to ACMG 2015 guidelines, showing 99.7% overall consistency with only 0.3% discrepancies. Differences in classifications between Dutch labs or between Dutch labs and ACMG were mainly present in genes with low penetrance or for late onset disorders and highlight limitations of the current 5‐tier classification system. The data sharing boosted the quality of DNA diagnostics in Dutch labs, an initiative we hope will be followed internationally. Recently, a positive match with a case from outside our consortium resulted in a more definite disease diagnosis.
To determine the genotype-phenotype association in patients with adenosine deaminase-2 (ADA2) deficiency due to identical homozygous R169Q mutations inCECR1 METHODS: We present a case series of nine ...ADA2-deficient patients with an identical homozygous R169Q mutation. Clinical and diagnostic data were collected and available MRI studies were reviewed. We performed genealogy and haplotype analyses and measured serum ADA2 activity. ADA2 activity values were correlated to clinical symptoms.
Age of presentation differed widely between the nine presented patients (range: 0 months to 8 years). The main clinical manifestations were (hepato)splenomegaly (8/9), skin involvement (8/9) and neurological involvement (8/9, of whom 6 encountered stroke). Considerable variation was seen in type, frequency and intensity of other symptoms, which included aplastic anaemia, acute myeloid leukaemia and cutaneous ulcers. Common laboratory abnormalities included cytopenias and hypogammaglobulinaemia. ADA2 enzyme activity in patients was significantly decreased compared with healthy controls. ADA2 activity levels tended to be lower in patients with stroke compared with patients without stroke. Genealogical studies did not identify a common ancestor; however, based on allele frequency, a North-West European founder effect can be noted. Three patients underwent haematopoietic cell transplantation, after which ADA2 activity was restored and clinical symptoms resolved.
This case series revealed large phenotypic variability in patients with ADA2 deficiency though they were homozygous for the same R169Q mutation inCECR1 Disease modifiers, including epigenetic and environmental factors, thus seem important in determining the phenotype. Furthermore, haematopoietic cell transplantation appears promising for those patients with a severe clinical phenotype.
With the introduction of Next Generation Sequencing (NGS) techniques increasing numbers of disease-associated variants are being identified. This ongoing progress might lead to diagnoses in formerly ...undiagnosed patients and novel insights in already solved cases. Therefore, many studies suggest introducing systematic reanalysis of NGS data in routine diagnostics. Introduction will, however, also have ethical, economic, legal and (psycho)social (ELSI) implications that Genetic Health Professionals (GHPs) from laboratories should consider before possible implementation of systematic reanalysis. To get a first impression we performed a scoping literature review. Our findings show that for the vast majority of included articles ELSI aspects were not mentioned as such. However, often these issues were raised implicitly. In total, we identified nine ELSI aspects, such as (perceived) professional responsibilities, implications for consent and cost-effectiveness. The identified ELSI aspects brought forward necessary trade-offs for GHPs to consciously take into account when considering responsible implementation of systematic reanalysis of NGS data in routine diagnostics, balancing the various strains on their laboratories and personnel while creating optimal results for new and former patients. Some important aspects are not well explored yet. For example, our study shows GHPs see the values of systematic reanalysis but also experience barriers, often mentioned as being practical or financial only, but in fact also being ethical or psychosocial. Engagement of these GHPs in further research on ELSI aspects is important for sustainable implementation.
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