In Vitro Analysis of the Functional Effects of an NLRP3 G809S Variant with the co-Existence of MEFV Haplotype Variants in Atypical Autoinflammatory Syndrome

Purpose

Hereditary periodic fever syndromes have been considered monogenic diseases. However, some recent reports have described patients with co-existence of recurrent fever responsible genes. This study assessed whether a rare variant, found in Japanese children showing atypical autoinflammatory syndrome, located in the leucine-rich repeat domain of Nod-like receptor family, pyrin domain containing 3 (NLRP3) with co-existence of Mediterranean fever (MEFV) haplotype variants may contribute to a proinflammatory phenotype using a systematic approach.

Methods

Cytokine production in serum or from peripheral blood monocytes was measured by ELISA. DNA sequence analysis of genes including NLRP3, MEFV, mevalonate kinase (MVK), and tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) were performed on patient samples. In vitro functional assays determined the effects of the NLRP3 variants and pyrin using NF-κB activation and speck formation assays.

Results

A heterozygous genetic variant of NLRP3, G809S, was found in samples from both patients. Additionally the previously reported heterozygous MEFV variants (P369S-R408Q or E148Q-P369S-R408Q) were also detected in both patients. Serum IL-1ra and sTNFR1 levels increased in the attack phase of the disease in both patients. The production levels of IL-1β from monocytes isolated from both cases were elevated following LPS and IFN-γ stimulation. The NLRP3 G809S variant demonstrated no increase of NF-κB activity following monosodium urate stimulation, whereas it significantly increased speck formation by interacting with apoptosis-associated speck-like protein with caspase recruitment domain.

Conclusions

The phenotype of atypical autoinflammatory disease in patients could be modified by a synergistic effect with two other variants of autoinflammatory-associated genes.     

An Atypical Outbreak of Food-Borne Botulism Due to Clostridium botulinum Types B and E from Ham

An outbreak of human botulism was due to consumption of ham containing botulinum neurotoxins B and E. A Clostridium botulinum type E strain isolated from ham was assigned to a new subtype (E12) based on bont/E gene sequencing and belongs to a new multilocus sequence subtype, as analyzed by whole-genome sequencing.     

An Atypical Ultrastructural Pattern in Fabry's Disease: A Study on Its Nature and Incidence in 7 Cases

This is a report on a stored lipid atypical ultrastructural pattern in skin samples of Fabry's disease expressed exclusively in the endothelium. The pattern consisted of intersecting short crescentic tightly packed membranes with a periodicity identical to that in classical ultrastructural variants. At low magnification the lysosomal aggregates of the material resembled “sunbursts” or aggregates of densely packed squirming villus-like structures. According to results of ultrastructural, lipid, and lectin histochemical analyses including analysis of the patients' blood groups, it could be concluded that it is just a variant physical state of the otherwise typical Fabry lipid. Its origin could be attributed to impeded formation (or maintenance) of larger lipid lamellae. It was found in great amounts in skin capillaries in 2 cases, and rarely in 5 additional cases. Knowledge of this atypical ultrastructural pattern is of practical significance because it could, if prevalent, cause diagnostic problems.     

An Atypical Ultrastructural Pattern in Fabry's Disease: A Study on Its Nature and Incidence in 7 Cases

This is a report on a stored lipid atypical ultrastructural pattern in skin samples of Fabry's disease expressed exclusively in the endothelium. The pattern consisted of intersecting short crescentic tightly packed membranes with a periodicity identical to that in classical ultrastructural variants. At low magnification the lysosomal aggregates of the material resembled “sunbursts” or aggregates of densely packed squirming villus-like structures. According to results of ultrastructural, lipid, and lectin histochemical analyses including analysis of the patients' blood groups, it could be concluded that it is just a variant physical state of the otherwise typical Fabry lipid. Its origin could be attributed to impeded formation (or maintenance) of larger lipid lamellae. It was found in great amounts in skin capillaries in 2 cases, and rarely in 5 additional cases. Knowledge of this atypical ultrastructural pattern is of practical significance because it could, if prevalent, cause diagnostic problems.     

Pulmonary Disease Due to Mycobacterium arosiense,an Easily Misidentified Pathogenic Novel Mycobacterium

Mycobacterium arosiense is a newly described species. After noticing it was misidentified as Mycobacterium intracellulare by the commercial identification system GenoType CM (Hein, Nehren, Germany), we detected 4 such strains among 33 that were previously misidentified as M. intracellulare. Three more strains were found among unidentified mycobacteria not tested previously with GenoType. The first case of pulmonary disease due to M. arosiense is reported here, and the novel species, of which so far only one strain had been investigated, is further characterized.A 62-year-old male, previously a smoker, was hospitalized because of a cough and slight fever (37.6°C). His history included Hodgkin''s lymphoma, treated with radiotherapy and a splenectomy at the age of 39, and gastric carcinoma, treated with a gastrectomy at the age of 53. In the last 5 months, the patient had been occasionally treated with amoxicillin-clavulanic acid; following each cycle, the fever and cough appeared to improve but invariably relapsed 15 to 20 days later. A chest X ray revealed bilateral fibronodular infiltration and calcified hilar nodules. A computed tomography scan showed, in the right apex of the lung, alveolar pseudonodular opacity and, in the upper left lobe, bronchiectasis and pleural thickening; typical tree-in-bud pictures (4), considered suggestive of mycobacterial infection, were present in the upper left lobe, both lower lobes, the middle lobe, and the lingula (Fig. ​(Fig.1).1). Both a skin test and a gamma interferon-releasing assay (8) for tuberculosis were negative. More-remarkable blood parameters included elevated erythrocyte sedimentation rate and C-reactive protein (104 mm/h and 1,590 μg/liter, respectively) and leukocytosis (16.4 × 10⁹ cells/liter) with neutrophilia (79%). When microbiological investigations of samples of sputum, urine, stool, blood, and pharyngeal swabs as well as serology for more-frequent viral and bacterial infections scored negative, the patient, who although untreated had turned afebrile, was discharged. Two weeks later, however, the growth on liquid MGIT medium (Becton Dickinson, Sparks, MD) of mycobacteria from four (out of four) sputum samples led to the start of an antituberculosis treatment with isoniazid, rifampin, pyrazinamide, and ethambutol. A new sample of sputum, collected at that time, grew mycobacteria 2 weeks later. Once the isolates were identified with GenoType CM (Hein, Nehren, Germany) (10) as belonging to the species Mycobacterium intracellulare, clarithromycin was added to the therapy, and isoniazid and pyrazinamide were removed. Since then, apart from episodes of epigastralgia and esophageal candidosis, the cough disappeared, and the conditions of the man clearly improved; the only sample of sputum he was able to produce remained negative in culture. At present, the patient has completed his fourth month of therapy, which is planned to be continued for at least 1 year (3), and is well. In the meantime, a thorough revision of the identification of the strain led to its being identified as belonging to the new species Mycobacterium arosiense.Open in a separate windowFIG. 1.Computed tomography scan showing a typical tree-in-bud picture.M. arosiense is a recently described, slow-growing, yellow-pigmented, and scotochromogenic species (2). The only strain reported so far was grown repeatedly from osteomyelitic bone lesions from a young boy with a hereditary partial gamma interferon receptor alpha-1 deficiency (2).We recently noticed that one strain identified by the reverse hybridization system GenoType as Mycobacterium intracellulare presented genetic sequences identical to the ones reported for M. arosiense. A thorough revision of the identification of the 33 strains assigned to M. intracellulare by GenoType revealed that 4 of them belonged to the new species M. arosiense. Such a finding allows us to hypothesize that M. arosiense is a mycobacterium that is less rare than it has been considered so far and adds clinical and microbiological information to the characterization of the new species, based thus far on a single strain.GenoType was performed on mycobacterial colonies subcultured on Middlebrook 7H11 agar, according to the recommendations of the producer (10).DNAs extracted from 33 strains isolated from independent clinical specimens, mostly respiratory materials, and identified as M. intracellulare by GenoType CM were initially submitted to genetic sequencing, following a previously reported protocol, of the internal transcribed spacer (ITS) region interposed between the 16S rRNA and the 23S rRNA genes (9). Four strains presenting ITS sequences identical to the one reported for M. arosiense were further sequenced by following the standard procedures and, in particular, were investigated for the almost complete 16S rRNA genes (5), a trait of about 400 bp of the gene coding for the 65-kDa heat shock protein (hsp65) (6) and a trait of about 700 bp of the gene coding for the beta subunit of RNA polymerase (rpoB) (1). A similar characterization was performed on three more strains isolated previously, when GenoType was not yet in use in our laboratory, and were considered, on the basis of partial sequencing of 16S rRNA genes, as not belonging to any known species (genetic sequences of M. arosiense were, at the time of their isolation, not yet available in GenBank).The susceptibility testing was performed, according to NCCLS recommendations (7), by determining the MICs for amikacin, ciprofloxacin, clarithromycin, ethambutol, gatifloxacin, linezolid, minocycline, moxifloxacin, rifabutin, rifampin, streptomycin, and trimethoprim-sulfamethoxazole using the commercially available microplates Sensititre MAISLOW (Trek Diagnostic Systems, Cleveland, OH).Clinical information was obtained from the patients'' records.The seven strains characterized here presented identical sequences, 100% overlapping the ones reported for M. arosiense, in the ITS region (GenBank/EMBL/DDBJ accession number {"type":"entrez-nucleotide","attrs":{"text":"EU370533","term_id":"169264491","term_text":"EU370533"}}EU370533) and in the 16S rRNA (GenBank/EMBL/DDBJ accession number {"type":"entrez-nucleotide","attrs":{"text":"EF054881","term_id":"172053330","term_text":"EF054881"}}EF054881), hsp65 (GenBank/EMBL/DDBJ accession number {"type":"entrez-nucleotide","attrs":{"text":"EU370531","term_id":"169264470","term_text":"EU370531"}}EU370531), and rpoB (GenBank/EMBL/DDBJ accession number {"type":"entrez-nucleotide","attrs":{"text":"EU350732","term_id":"164459579","term_text":"EU350732"}}EU350732) genes.They were all slow growers and presented scotochromogenic yellow pigmentation. Most of them had grown, in primary cultures, only in liquid media, although in subcultures, they were also able to grow on solid media (both egg and agar based).The antimicrobial pattern (Table ​(Table1)1) was very homogeneous: all the strains were resistant to quinolones, ethambutol, and streptomycin and susceptible to rifamycins and clarithromycin. They were moderately susceptible to amikacin and linezolid and moderately resistant to minocycline.

TABLE 1.

MICs for M. arosiense strains^a

A CLAG3 Mutation in an Amphipathic Transmembrane Domain Alters Malaria Parasite Nutrient Channels and Confers Leupeptin Resistance

Erythrocytes infected with malaria parasites have increased permeability to ions and nutrients, as mediated by the plasmodial surface anion channel (PSAC) and recently linked to parasite clag3 genes. Although the encoded protein is integral to the host membrane, its precise contribution to solute transport remains unclear because it lacks conventional transmembrane domains and does not have homology to ion channel proteins in other organisms. Here, we identified a probable CLAG3 transmembrane domain adjacent to a variant extracellular motif. Helical-wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of a predicted α-helical transmembrane domain, suggesting that the domain lines a water-filled pore. A single CLAG3 mutation (A1210T) in a leupeptin-resistant PSAC mutant falls within this transmembrane domain and may affect pore structure. Allelic-exchange transfection and site-directed mutagenesis revealed that this mutation alters solute selectivity in the channel. The A1210T mutation also reduces the blocking affinity of PSAC inhibitors that bind on opposite channel faces, consistent with global changes in channel structure. Transfected parasites carrying this mutation survived a leupeptin challenge significantly better than a transfection control did. Thus, the A1210T mutation contributes directly to both altered PSAC activity and leupeptin resistance. These findings reveal the molecular basis of a novel antimalarial drug resistance mechanism, provide a framework for determining the channel''s composition and structure, and should guide the development of therapies targeting the PSAC.     

Identification of a de novo NLRP3 gene variation in an Italian Behçet syndrome patient

A novel nonsynonymous variation of NLRP3 was identified in an Italian patient with Behçet syndrome using both bioinformatics and molecular methods. This variation was a thymine to guanine polymorphism responsible for the isoleucine to serine amino acid change at position 348. The novel variation was predicted to be a pathogenic allele.     

Massively Parallel Sequencing Detected a Mutation in the MFN2 Gene Missed by Sanger Sequencing Due to a Primer Mismatch on an SNP Site

We describe a patient with early onset severe axonal Charcot‐Marie‐Tooth disease (CMT2) with dominant inheritance, in whom Sanger sequencing failed to detect a mutation in the mitofusin 2 (MFN2) gene because of a single nucleotide polymorphism (rs2236057) under the PCR primer sequence. The severe early onset phenotype and the family history with severely affected mother (died after delivery) was very suggestive of CMT2A and this suspicion was finally confirmed by a MFN2 mutation. The mutation p.His361Tyr was later detected in the patient by massively parallel sequencing with a gene panel for hereditary neuropathies. According to this information, new primers for amplification and sequencing were designed which bind away from the polymorphic sites of the patient's DNA. Sanger sequencing with these new primers then confirmed the heterozygous mutation in the MFN2 gene in this patient. This case report shows that massively parallel sequencing may in some rare cases be more sensitive than Sanger sequencing and highlights the importance of accurate primer design which requires special attention.     

MEFV and NLRP3 Inflammasome Expression Is Attributed to Immature Macrophages and Correlates with Serum Inflammatory Proteins in Crohn´s Disease Patients

Inflammation - Inflammasomes are intracellular protein complexes whose activation results in proinflammatory cytokines. Inflammasomes are implicated in Crohn´s disease (CD) pathogenesis, yet...     

An Overview and Update of ATP7A Mutations Leading to Menkes Disease and Occipital Horn Syndrome

Menkes disease (MD) is a lethal multisystemic disorder of copper metabolism. Progressive neurodegeneration and connective tissue disturbances, together with the peculiar “kinky” hair, are the main manifestations. MD is inherited as an X‐linked recessive trait, and as expected the vast majority of patients are males. MD occurs because of mutations in the ATP7A gene and the vast majority of ATP7A mutations are intragenic mutations or partial gene deletions. ATP7A is an energy‐dependent transmembrane protein, which is involved in the delivery of copper to the secreted copper enzymes and in the export of surplus copper from cells. Severely affected MD patients die usually before the third year of life. A cure for the disease does not exist, but very early copper–histidine treatment may correct some of the neurological symptoms. This study reviews 274 published and 18 novel disease causing mutations identified in 370 unrelated MD patients, nonpathogenic variants of ATP7A, functional studies of the ATP7A mutations, and animal models of MD.     

Autosomal Recessive Agammaglobulinemia Due to a Homozygous Mutation in <Emphasis Type="Italic">PIK3R1</Emphasis>

The role of class IA phosphoinositide 3 kinases (PI3Ks) in immune function and regulation continues to expand with the identification of greater numbers of genetic variants. This case report is the second reported case of a homozygous premature stop codon within the PIK3R1 gene leading to autosomal recessive agammaglobulinemia. The proband, born to consanguineous parents, presented at 10 months of age with a history of oropharyngeal petechiae and bleeding from the mouth, gums, and tear ducts. Initial investigations revealed thrombocytopenia, neutropenia and the absence of B cells. Further genetic testing via a custom next-generation sequencing panel confirmed the presence of a homozygous mutation in PIK3R1, c.901 C>T, a premature stop codon at amino acid position 301. Given their many roles in immune regulation, recessive mutations in the PlK3R1 gene should be considered in infants presenting with hypogammaglobulinemia or agammaglobulinemia, particularly in the setting of parental consanguinity.     

Case of cyclopia with an unbalanced karyotype attributable to a balanced 3/7 translocation

This report presents a case of cyclopia attributable to an unbalanced karyotype in a family with a balanced, reciprocal 3/7 translocation. This case was the fifth recorded in three generations of this family. From this report it is possible that the simultaneous action of partial trisomy 3p and partial monosomy 7q may be one cause of holoprosencephaly.     

Binding of [3H]ICIA 5165, an H2-receptor antagonist to guinea pig gastric mucosa

ICIA 5165, 2-guanidino-4-[4-(2-cyano-3-methylguanidino)butyl] thiazole, a selective histamine H₂-receptor antagonist was radiolabelled with tritium to a specific activity of 50.8 Ci/mmoll for use in binding studies. Radiolabelling did not impair bioactivity.Binding characteristics of [³H]ICIA 5165 to guinea pig gastric mucosa were determined. Ligand binding was rapid, reaching equilibrium within five minutes at 0°C, reversible and saturable. Specific [³H]ICIA 5165 binding had an equilibrium dissociation constant of 1.29×10^–8 M, determined by Scatchard plot analysis, and of 1.02×10^–8 M, calculated from the ratio of the dissociation to association rate constants. A Hill number, n_H, of 1.02 was determined for the specific binding component. Specific binding of [³H]ICIA 5165 to gastric mucosal supernatant was not inhibited by methapyrilene, diphenhydramine, mepyramine, d-chlorpheniramine or I-chlorpheniramine (all at 10^–7 M), or by atropine or propranolol (both at 10^–6 M). Specific [³H]ICIA 5165 binding was inhibited in a concentration dependent manner by non-radioactive ICIA 5165 and tiotidine, as well as by a variety of other agents, with H₂ agonist or H₂ antagonist properties. In competition experiments, however, difficulties encountered in accurately defining the degree of specific binding indicate some reservation should be observed in interpreting these results.     

HLA-DR3- and HLA-DR7-Restricted T-Cell Hyporesponsiveness to Gluten Antigen: A Clue to the Aetiology of Coeliac Disease?

Coeliac disease (CD) is strongly associated with the human class-II HLA determinants HLA-DR3 and -DR7. We investigated the relative frequency of gluten-reactive T cells from DR3- or DR7-positive. CD patients and healthy controls who were heterozygous at the DR locus. We found a consistently and significantly lower frequency of gluten-reactive T cells when the antigen was presented by monocytes in conjunction with DR3 or DR7 than in conjunction with the other DR determinant of the T-cell donor. In contrast, the frequency of reactive T cells in these donors to other antigens was not reduced in conjunction with DR3 or DR7. These results indicate a specific immunoregulatory function associated with class-II HLA molecules. The reduced frequency of gluten-reactive T cells in association with HLA-DR3 or -DR7 may be directly involved in the development of CD.