A new category of autoinflammatory disease associated with NOD2 gene mutations

Introduction

Autoinflammatory diseases are characterized by seemingly unprovoked episodes of inflammation, without high titers of autoantibodies or antigen-specific T cells, and derive from genetic variants of the innate immune system. This study characterized a cohort of patients with similar phenotypes and nucleotide oligomerization domain 2 (NOD2) gene mutations.

Methods

Diagnostically challenging patients with the following clinical and genetic characteristics were prospectively studied between January 2009 and April 2011: periodic fever, dermatitis, polyarthritis, serositis, negative serum autoantibodies and additional positive NOD2 IVS8⁺¹⁵⁸ gene mutation. Genetic testing for gene mutations of NOD2, tumor necrosis factor receptor-associated periodic fever syndrome (TRAPS) and familial Mediterranean fever (FMF) was performed.

Results

All seven patients with the disease were Caucasians, with four being male. The mean age at disease onset was 40.7 years and disease duration was 3.2 years. These patients characteristically presented with periodic fever, dermatitis and inflammatory polyarthritis. There were gastrointestinal symptoms in three patients, granulomas of the skin and gut in two, and recurrent chest pain in two, with one having pleuritis and pericarditis. Three patients had sicca-like symptoms. Five patients had increased acute phase reactants. All seven patients had negative tests for autoantibodies but carried the NOD2 gene mutation IVS8⁺¹⁵⁸ with four having concurrent R702W mutation.

Conclusions

Our cohort may represent a new disease category of autoinflammatory disease with characteristic clinical phenotypes and genotypes. It may somewhat resemble pediatric Blau's syndrome.     

Effects of peroxynitrite on sarco/endoplasmic reticulum Ca2+ pump isoforms SERCA2b and SERCA3a

Sarco(endo)plasmic reticulum Ca²⁺ (SERCA) pumps are important for cell signaling. Three different genes, SERCA1, 2, and 3, encode these pumps. Most tissues, including vascular smooth muscle, express a splice variant of SERCA2 (SERCA2b), whereas SERCA3a is widely distributed in tissues such as vascular endothelium, tracheal epithelium, mast cells, and lymphoid cells. SERCA2b protein is readily inactivated by peroxynitrite that may be formed during cardiac ischemia reperfusion or during immune response after infection. Here, we compared the peroxynitrite sensitivity of SERCA2b and SERCA3a by using microsomes prepared from HEK-293T cells overexpressing the pumps. We incubated the microsomes with different concentrations of peroxynitrite and determined Ca²⁺ uptake, Ca²⁺-Mg²⁺-ATPase, Ca²⁺-dependent formation of acylphosphate intermediate, and protein mobility in Western blots. Ca²⁺ uptake, Ca²⁺-Mg²⁺-ATPase, and Ca²⁺-dependent formation of acylphosphate intermediate were inactivated for both SERCA2b and SERCA3a, but the latter was more resistant to the inactivation. Western blots showed that SERCA2b and SERCA3a proteins oligomerized after treatment with peroxynitrite, but each with a slightly different pattern. Compared with monomers, the oligomers may be less efficient in forming the acylphosphate intermediate and in conducting the remainder of the steps in the reaction cycle. We conclude that the resistance of SERCA3a to peroxynitrite may aid the cells expressing them in functioning during exposure to oxidative stress. free radicals; Ca²⁺-Mg²⁺-ATPase; ischemia; coronary artery; vascular smooth muscle; sarco(endo)plasmic reticulum Ca²⁺ pumps     

Ca2+-dependent redox modulation of SERCA 2b by ERp57

We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.     

Clinical case of acral hemorrhagic Darier's disease is not caused by mutations in exon 15 of the ATP2A2 gene

Darier's disease (Dyskeratosis follicularis, DD) is a genetic disorder characterized by pathogenetic changes of keratinization with variant forms of cutaneous phenotype. Recently, it has been showed that Darier's disease cause mutations in the ATP2A2 gene, at 12q24.1. The gene encodes sarco-endoplasmic reticulum calcium ATPase type 2 (SERCA2). Mutations in exon 15 are reported to be the most consistent mutations associated with the acral hemorrhagic type of Darier's disease. By direct sequencing we investigated exon 15 of the ATP2A2 gene in a Croation family in which one member had a hemorrhagic Darier's disease, but did not record any mutation in the family we investigated. Our results show that mutations in exon 15 of the ATP2A2 gene are not a necessary prerequisite for acral hemorrhagic type of Darier's disease. Our finding support the variability of clinical manifestations of Darier's disease and lack of genotype/phenotype consistency.     

Multiple mitochondrial tRNAMLeu[UUR] mutations associated with infantile myopathy

We have identified a cluster of mitochondrial tRNA^Leu[UUR], mutations in a severe case of infantile myopathy. There were A to G transitions found at mtDNA positions 3259, 3261, 3266 and 3268. These point mutations change the anticodon arm and the anticodon UAA, normally found in tRNA^Leu[UUR], to UGA which is the one of the tRNAs^Ser[UCN]. This is the first anticodon alteration described in this tRNA. Another swap straight to the anticodon of tRNA^Pro alone was recently described in a less severe case [1]. Until now infantile myopathies have not been attributed to defined mtDNA alterations. This study reports for the first time mtDNA point mutations causing this early onset of a mitochondrial disorder. The apparent homoplasmy of these mutations and especially the location in the anticodon must be considered lethal, if the child would not have been respirated for 5 years from its birth. (Mol Cell Biochem 174: 231–236, 1997)     

Two de novo mutations of MFN2 associated with early-onset Charcot-Marie-Tooth disease type 2A neuropathy

Charcot-Marie-Tooth disease type 2A (CMT2A) is one of the subdivisions of CMT2, an axonal defective form of peripheral neuropathy. Different mutations in the mitochondrial GTPase mitofusin 2 (MFN2) gene produce various degrees of severity of CMT2A phenotype or CMT2A related hereditary motor and sensory neuropathy VI (HMSN VI). The occurrence of de novo mutations in MFN2 is by far the most frequent as compared to other CMT genes. About 26% of the pathogenic MFN2 mutations reported in the Inherited Peripheral Neuropathies Mutations Database are de novo. This study identified two de novo mutations of MFN2, c.1048T>C (S350P) and c.310C>T (R104W), from two Korean CMT2A patients with early onset severe clinical symptoms. The comparative genotype-phenotype correlations of these mutations according to a previously reported case were also viewed. The R104W mutation has been reported recurrently, outspread over different ethnic backgrounds as de novo. The re-occurrence of the same pathogenic de novo variants within and amongst different ethnic groups clearly suggests a susceptible hot spot for mutation in the MFN2 gene. If the deleterious mutations discourage fitness and reproduction, this negative selection factor should ultimately reduce the prevalence of the disease. It appears that spontaneous de novo mutations in turn seem to be maintaining the disease phenotype??s prevalence.     

Ca2+ transport ATPase isoforms SERCA2a and SERCA2b are targeted to the same sites in the murine heart

Adult SERCA2(b/b) mice expressing the non-muscle Ca2+ transport ATPase isoform SERCA2b in the heart instead of the normally predominant sarcomeric SERCA2a isoform, develop mild concentric ventricular hypertrophy with impaired cardiac contractility and relaxation [Circ. Res. 89 (2001) 838]. Results from a separate study on transgenic mice overexpressing SERCA2b in the normal SERCA2a context were interpreted to show that SERCA2b and SERCA2a are differentially targeted within the cardiac sarcoplasmic reticulum (SR) [J. Biol. Chem. 275 (2000) 24722]. Since a different subcellular distribution of SERCA2b could underlie alterations in Ca2+ handling observed in SERCA2(b/b), we wanted to compare SERCA2b distribution in SERCA2(b/b) with that of SERCA2a in wild-type (WT). Using confocal microscopy on immunostained fixed myocytes and BODIPY-thapsigargin-stained living cells, we found that in SERCA2(b/b) mice SERCA2b is correctly targeted to cardiac SR and is present in the same SR regions as SERCA2a and SERCA2b in WT. We conclude that there is no differential targeting of SERCA2a and SERCA2b since both are found in the longitudinal SR and in the SR proximal to the Z-bands. Therefore, alterations in Ca2+ handling and the development of hypertrophy in adult SERCA2(b/b) mice do not result from different SERCA2b targeting.     

Sarco/endoplasmic reticulum Ca2+-pump isoform SERCA3a is more resistant to superoxide damage than SERCA2b

Endo/sarcoplasmic reticulum (ER) Ca²⁺-pumps are important for cell survival and communication but they are inactivatedby reactive oxygen species (ROS).We have previously reported that the Ca²⁺-pump isoform SERCA3a is more resistant than SERCA2b to damage by peroxide. Since peroxide and superoxide differ in their redox potentials, we now report the effects of superoxide on the two Ca²⁺-pump isoforms. We isolated microsomes from HEK293 cells transiently transfected with SERCA2b or SERCA3a cDNA. We exposed these microsomes to superoxide which was generated using xanthine plus xanthine oxidase and catalase to prevent accumulation of peroxide due to superoxide dismutation. Superoxide damaged the Ca²⁺- transport activity of both isoforms but SERCA3a was damaged at higher concentrations of superoxide and upon longer periods of exposures than was SERCA2b. Thus the SERCA3a isoform is more resistant than SERCA2b to inactivation by both superoxide and peroxide. (Mol Cell Biochem 000: 000-000, 1999)     

ER‐to‐Golgi blockade of nascent desmosomal cadherins in SERCA2‐inhibited keratinocytes: Implications for Darier's disease

Darier's disease (DD) is an autosomal dominantly inherited skin disorder caused by mutations in sarco/endoplasmic reticulum Ca²⁺‐ATPase 2 (SERCA2), a Ca²⁺ pump that transports Ca²⁺ from the cytosol to the endoplasmic reticulum (ER). Loss of desmosomes and keratinocyte cohesion is a characteristic feature of DD. Desmosomal cadherins (DC) are Ca²⁺‐dependent transmembrane adhesion proteins of desmosomes, which are mislocalized in the lesional but not perilesional skin of DD. We show here that inhibition of SERCA2 by 2 distinct inhibitors results in accumulation of DC precursors in keratinocytes, indicating ER‐to‐Golgi transport of nascent DC is blocked. Partial loss of SERCA2 by siRNA has no such effect, implicating that haploinsufficiency is not sufficient to affect nascent DC maturation. However, a synergistic effect is revealed between SERCA2 siRNA and an ineffective dose of SERCA2 inhibitor, and between an agonist of the ER Ca²⁺ release channel and SERCA2 inhibitor. These results suggest that reduction of ER Ca²⁺ below a critical level causes ER retention of nascent DC. Moreover, colocalization of DC with ER calnexin is detected in SERCA2‐inhibited keratinocytes and DD epidermis. Collectively, our data demonstrate that loss of SERCA2 impairs ER‐to‐Golgi transport of nascent DC, which may contribute to DD pathogenesis.     

Cytosolic phosphorylation of calnexin controls intracellular Ca(2+) oscillations via an interaction with SERCA2b

Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca(2+) imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca(2+) oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate- mediated Ca(2+) release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca(2+) signaling and controlling Ca(2+)-sensitive chaperone functions in the ER.     

Assessing environmental modifiers of disease risk associated with rare mutations

OBJECTIVE: As disease-predisposing mutations are increasingly identified, there is growing need to assess the effects of lifestyle and environmental factors on disease risks in mutation carriers. Such assessment is difficult when the mutations are rare and evaluating them in large population samples is costly. METHODS: This paper describes four study designs for evaluating the effects of environmental exposures in carriers of rare disease-predisposing mutations. RESULTS: The strengths and weaknesses of the designs are assessed, and strategies for analyzing the data obtained from such designs are considered. CONCLUSIONS: When exposure effects in noncarriers are well-established and exposure is independent of carrier status in the population of disease-free controls, the case-only design provides a feasible and efficient method for inferring effects in carriers. When exposure effects in noncarriers are not well established, the most feasible design options are those that compare exposures in carrier cases to either untyped controls or to carrier controls. These two designs have complementary strengths and weaknesses; thus inferences are stronger when measures of association estimated using the two designs are consistent.     

Two cloned β thalassemia genes are associated with amber mutations at codon 39

Two β globin genes from patients with the β⁺ thalassemia phenotype have been cloned and sequenced. A single nucleotide change from CAG to TAG (an amber mutation) at codon 39 is the only difference from normal in both genes analyzed. The results are consistent with the assumption that both patients are doubly heterozygous for β⁺ and β° thalassemia, and that we have isolated and analyzed the β° thalassemia gene.     

Multiple endocrine neoplasia associated with von Recklinghausen's disease.

Details were studied of three patients with duodenal carcinoid tumour in association with neurofibromatosis and phaeochromocytoma, and of four patients with duodenal carcinoid and either von Recklinghausen''s disease or phaeochromocytoma. The rarity of these endocrine tumours, together with the unusual morphological features and somatostatin content of the two duodenal carcinoids examined, suggest that this combination of tumours is not a chance association. It is suggested that this linkage of neurofibromatosis, phaeochromocytoma, and duodenal carcinoid is a specific multiple endocrine neoplasia syndrome.     

Clinical atovaquone-proguanil resistance of Plasmodium falciparum associated with cytochrome b codon 268 mutations

Plasmodium falciparum resistance to atovaquone-proguanil has so far been associated with Y268S or Y268N mutations in cytochrome b, although these changes were identified in only seven of the 11 treatment failures. Here, we describe 10 new cases of atovaquone-proguanil treatment failures among which the parasite resistance was confirmed in six cases, either by identifying correct plasma drug concentrations or by observing in vitro atovaquone resistance. Resistance was consistently associated with codon 268 mutations (Y268S or a previously unidentified mutation, Y268C). Notably, mutations were not detected before the treatment but only after the drug exposure.     

Lnc’ing Ca2+, SERCA and cardiac disease

Loss of SERCA function contributes to reduced contractility, Ca²⁺ overload and arrhythmias in the diseased heart. A long non-coding RNA (ZFAS1) upregulated in cardiac disease is reported to directly inhibit SERCA function. The implications for cardiac disease and the wider roles of SERCA are discussed.     

Wilson disease mutations associated with uncommon haplotypes in mediterranean patients

This study reports 12 novel mutations of the Wilson disease (WD) gene which have been detected by the molecular analysis of 29 patients of Mediterranean descent carrying uncommon chromosomal haplotypes at the WD locus. These mutations include two nonsense, one splice site and nine missense. The missense mutations lie in regions of the WD gene critical for its function, such as the transmembrane region, the transduction domain and the ATP loop and ATP-binding domain, indicating that they are disease-causing mutations. These new findings improve our knowledge for the role played by functional domains on the ATP7B function. Received: 20 March 1996