Characterization of Novel Pathogenic Variants Leading to Caspase-8 Cleavage-Resistant RIPK1-Induced Autoinflammatory Syndrome.

Pathogenic RIPK1 variants have been described as the cause of two different inborn errors of immunity. Biallelic loss-of-function variants cause the recessively inherited RIPK1 deficiency, while monoallelic variants impairing the caspase-8-mediated RIPK1 cleavage provoke a novel autoinflammatory disease (AID) called cleavage-resistant RIPK1-induced autoinflammatory (CRIA) syndrome. The aim of this study was to characterize the pathogenicity of two novel RIPK1 variants located at the cleavage site of caspase-8 detected in patients with dominantly-inherited, early-onset undefined AID. RIPK1 genotyping was performed by Sanger and next-generation sequencing. Clinical and analytical data were collected from medical charts, and in silico and in vitro assays were performed to evaluate the functional consequences. Genetic analyses identified two novel heterozygous RIPK1 variants at the caspase-8 cleavage site (p.Leu321Arg and p.Asp324Gly), which displayed a perfect intrafamilial phenotype-genotype segregation following a dominant inheritance pattern. Structural analyses suggested that these variants disrupt the normal RIPK1 structure, probably making it less accessible to and/or less cleavable by caspase-8. In vitro experiments confirmed that the p.Leu321Arg and p.Asp324Gly RIPK1 variants were resistant to caspase-8-mediated cleavage and induced a constitutive activation of necroptotic pathway in a similar manner that previously characterized RIPK1 variants causing CRIA syndrome. All these results strongly supported the pathogenicity of the two novel RIPK1 variants and the diagnosis of CRIA syndrome in all enrolled patients. Moreover, the evidences here collected expand the phenotypic and genetic diversity of this recently described AID, and provide interesting data about effectiveness of treatments that may benefit future patients.

Case report: Novel compound heterozygous IL1RN mutations as the likely cause of a lethal form of deficiency of interleukin-1 receptor antagonist.

Undiagnosed monogenic diseases represent a challenging group of human conditions highly suspicious to have a genetic origin, but without conclusive evidences about it. We identified two brothers born prematurely from a non-consanguineous healthy couple, with a neonatal-onset, chronic disease characterized by severe skin and bone inflammatory manifestations and a fatal outcome in infancy. We conducted DNA and mRNA analyses in the patients' healthy relatives to identify the genetic cause of the patients' disease. DNA analyses were performed by both Sanger and next-generation sequencing, which identified two novel heterozygous IL1RN variants: the intronic c.318 + 2T>G variant in the father and a ≈2,600-bp intragenic deletion in the mother. IL1RN mRNA production was markedly decreased in both progenitors when compared with healthy subjects. The mRNA sequencing performed in each parent identified two novel, truncated IL1RN transcripts. Additional experiments revealed a perfect intrafamilial phenotype-genotype segregation following an autosomal recessive inheritance pattern. The evidences shown here supported for the presence of two novel loss-of-function (LoF) IL1RN pathogenic variants in the analyzed family. Biallelic LoF variants at the IL1RN gene cause the deficiency of interleukin-1 receptor antagonist (DIRA), a monogenic autoinflammatory disease with marked similarities with the patients described here. Despite the non-availability of the patients' samples representing the main limitation of this study, the collected evidences strongly suggest that the patients described here suffered from a lethal form of DIRA likely due to a compound heterozygous genotype at IL1RN, thus providing a reliable genetic diagnosis based on the integration of old medical information with currently obtained genetic data.

Application of a split-Cre system for high-capacity adenoviral vector amplification.

BACKGROUND AND AIMS: High-capacity adenoviral vectors (HC-AdV) show extended DNA payload and stability of gene expression in vivo due to the absence of viral coding sequences. However, production requires methods to trans-complement viral proteins, usually through Helper Viruses (HV). The Cre/loxP system is frequently employed to remove the packaging signal in HV genomes, in order to avoid their encapsidation. However, chronic exposure to the Cre recombinase in packaging cells is detrimental. We have applied the dimerizable Cre system to overcome this limitation. METHODS AND RESULTS: Cre was split in two fragments devoid of recombinase function (N-terminal 244 and C-terminal 99 amino-acids). In one version of the system, interaction with both moieties was favored by rapamycin-dependent heterodimerization domains (DiCre). Other version contained only Cre sequences (oCre). We generated packaging cells and HVs expressing the complementary fragments and studied their performance for HC-AdV production. We found that both conformations avoided interference with the growth of packaging cells, and the oCre system was particularly suitable for HC-AdV amplification. CONCLUSIONS: The split-Cre system improves the performance of packaging cells and can reduce the time and cost of HC-AdV amplification up to 30% and 15%, respectively. This may contribute to the standardization of HC-AdV production.

Spectrum of Disease Manifestations in Patients with Selective Immunoglobulin E Deficiency.

BACKGROUND: Selective IgE deficiency (SIgED) has been previously evaluated in selected patients from allergy units. This study investigates the effects of SIgED on the entire population in a hospital setting and sought to delineate in detail the clinical aspects of SIgED. METHODS: A retrospective study of the data obtained from electronic medical records of 52 adult patients (56% female) with a mean age of 43 years and IgE levels of <2.0 kU/L with normal immunoglobulin (Ig) IgG, IgA, and IgM levels, seen at our hospital, without selection bias, from 2010 to 2019. RESULTS: Recurrent upper respiratory infections were recorded in 18 (34.6%) patients, pneumonia was recorded in 16 (30.7%) patients, bronchiectasis was recorded in 16 (30.7%) patients, and asthma was recorded in 10 (19.2%) patients. Eighteen patients (34.6%) suffered autoimmune clinical manifestations either isolated (19%) or combining two or more diseases (15%), Hashimoto's thyroiditis being the most frequent (19%), which was followed by arthritis (10%) and thrombocytopenia and/or neutropenia (5.7%). Other less frequent associations were Graves' disease, primary sclerosing cholangitis, Sjögren's syndrome, and autoimmune hepatitis. Eczematous dermatitis (15.3%), chronic spontaneous urticaria (17.3%), and symptoms of enteropathy (21%) were also highly prevalent. Thirty percent of patients developed malignancies, with non-Hodgkin lymphomas (13.4%) being the most prevalent. CONCLUSIONS: The clinical manifestations of SIgED encompass a variety of infectious, non-infectious complications, and malignancy. Since it cannot be ruled out that some type of selection bias occurred in the routine assessment of IgE serum Ievels, prospective studies are required to better characterize SIgED and to determine whether it should be added to the list of antibody deficiencies.