IgE-binding to vicilin-like antimicrobial peptides is associated with systemic reactions to macadamia nut.

BACKGROUND: Macadamia nut can induce fatal allergic reactions and changes in dietary habits will raise their consumption in industrialised countries. Until now diagnosis of macadamia nut allergy by sIgE solely relies on the macadamia nut extract, but single components are lacking. METHODS: Macadamia nut proteins recognised by IgE from 2 macadamia nut extract positive sera were identified by mass spectrometry (vicilin-like antimicrobial peptides: VLAP). Sensitisation to macadamia nut extract and heterologously expressed isoform VLAP-2-3 was evaluated in 82 nut allergic (NA) and 27 tolerant (NT) patients (no tree nut allergy reported) comprehending 10 macadamia nut allergic (MA) and 18 explicitly reported macadamia nut tolerant patients (MT), using line blots. Co-sensitisation to additional VLAP isoforms and other vicilins was evaluated in 8 MA, 12 MT and 14 NA patients sensitised to VLAP-2-3. Functional properties were determined by indirect basophil activation. RESULTS: Even though proteins recognised by IgE were identified as VLAP-2-1, 2-2 and 2-3, only peptides specifically belonging to VLAP-2-3 were detected by mass spectrometry. The macadamia nut extract was recognised by 33% of NA patients (27/82) including 3 MA patients and 26% of NT patients (7/27, 3 MT). Similarly, 29% of NA (24/82) patients showed partly strong sIgE-binding to VLAP-2-3 including 3 MA patients with systemic reactions to macadamia nut. Contrary, VLAP-2-3 was recognised by only 2 NT (1 MT) patients (7%) with very low sIgE titres. Simultaneous recognition of the isoforms VLAP-2-1 and 2-2 was observed in all patients positive for VLAP-2-3 with partly reduced sIgE titres in 59% of these patients. Additionally, all three VLAP isoforms were able to repeatedly induce BAT reactivity upon sensitisation with a MA serum. CONCLUSION: VLAP proteins are the first described macadamia nut components with serological and functional allergenic properties and they are associated with systemic reactions to macadamia nut.

Sesame oleosins are minor allergens.

BACKGROUND: In daily practice, one-third of sesame allergic patients, confirmed by clinical history or food challenge, do not show any detectable specific IgE using current diagnostics. Currently used sesame extracts are water-based and therefore lacking hydrophobic proteins like oleosins. Oleosins, the stabilizer of lipid droplets in plants, are described as allergens in sesame, peanut and hazelnut. In this study, we examine the role of oleosins in sesame allergy and their potential cross-reactivity between sesame and (pea)nuts. METHODS: Specific IgE and IgG sensitisation to native and heterologously expressed sesame components and oleosins from other nuts, free of seed storage proteins, was assessed by line blot and sera from 17 sesame allergic patients without detectable specific IgE sensitisation to sesame, and compared to 18 sesame allergic and 13 tolerant patients with specific IgE sensitisation to sesame. RESULTS: Sesame allergic patients without sensitisation showed no specific IgE to the tested sesame oleosins or components. Low levels of specific IgE to sesame oleosins were detected in 17% of sesame allergic and 15% of tolerant patients with sIgE sensitisation. Oleosins were recognised by serum IgG from multiple patients confirming immune reactivity and excluding technical issues leading to lack of specific IgE-binding to oleosins. CONCLUSION: Sesame oleosins are minor allergens and appear to have no additonal value in diagnosing sesame allergy in adults based on sIgE and sIgG detection. There is a high need for additional diagnostic tools in those patients to minimize the number of required food challenges.

A positive genotype-phenotype correlation in a large cohort of patients with Pseudohypoparathyroidism Type Ia and Pseudo-pseudohypoparathyroidism and 33 newly identified mutations in the GNAS gene.

Maternally inherited inactivating GNAS mutations are the most common cause of parathyroid hormone (PTH) resistance and Albright hereditary osteodystrophy (AHO) leading to pseudohypoparathyroidism type Ia (PHPIa) due to Gsα deficiency. Paternally inherited inactivating mutations lead to isolated AHO signs characterizing pseudo-pseudohypoparathyroidism (PPHP). Mutations are distributed throughout the Gsα coding exons of GNAS and there is a lack of genotype-phenotype correlation. In this study, we sequenced exon 1-13 of GNAS in a large cohort of PHPIa- and PPHP patients and identified 58 different mutations in 88 patients and 27 relatives. Thirty-three mutations including 15 missense mutations were newly discovered. Furthermore, we found three hot spots: a known hotspot (p.D190MfsX14), a second at codon 166 (p.R166C), and a third at the exon 5 acceptor splice site (c.435 + 1G>A), found in 15, 5, and 4 unrelated patients, respectively. Comparing the clinical features to the molecular genetic data, a significantly higher occurrence of subcutaneous calcifications in patients harboring truncating versus missense mutations was demonstrated. Thus, in the largest cohort of PHPIa patients described to date, we extend the spectrum of known GNAS mutations and hot spots and demonstrate for the first time a correlation between the genetic defects and the expression of a clinical AHO-feature.

Evidence of hormone resistance in a pseudo-pseudohypoparathyroidism patient with a novel paternal mutation in GNAS.

CONTEXT: Loss-of-function GNAS mutations lead to hormone resistance and Albright's hereditary osteodystrophy (AHO) when maternally inherited, i.e. pseudohypoparathyroidism-Ia (PHPIa), but cause AHO alone when located on the paternal allele, i.e. pseudoPHP (PPHP). OBJECTIVE: We aimed to establish the molecular diagnosis in a patient with AHO and evidence of hormone resistance. CASE: The patient is a female who presented at the age of 13.5years with short stature and multiple AHO features. No evidence for TSH or gonadotropin-resistance was present. Serum calcium and vitamin D levels were normal. However, serum PTH was elevated on multiple occasions (64-178pg/mL, normal: 9-52) and growth hormone response to clonidine or L-DOPA was blunted, suggesting hormone resistance and PHP-Ia. The patient had diminished erythrocyte Gsα activity and a novel heterozygous GNAS mutation (c.328 G>C; p.A109P). The mother lacked the mutation, and the father's DNA was not available. Hence, a diagnosis of PPHP also appeared possible, supported by low birth weight and a lack of AHO features associated predominantly with PHP-Ia, i.e. obesity and cognitive impairment. To determine the parental origin of the mutation, we amplified the paternally expressed A/B and biallelically expressed Gsα transcripts from the patient's peripheral blood RNA. While both wild-type and mutant nucleotides were detected in the Gsα amplicon, only the mutant nucleotide was present in the A/B amplicon, indicating that the mutation was paternal. CONCLUSION: These findings suggest that PTH and other hormone resistance may not be an exclusive feature of PHP-Ia and could also be observed in patients with PPHP.

European guidance for the molecular diagnosis of pseudohypoparathyroidism not caused by point genetic variants at GNAS: an EQA study.

Pseudohypoparathyroidism is a rare endocrine disorder that can be caused by genetic (mainly maternally inherited inactivating point mutations, although intragenic and gross deletions have rarely been reported) or epigenetic alterations at GNAS locus. Clinical and molecular characterization of this disease is not that easy because of phenotypic, biochemical and molecular overlapping features between both subtypes of the disease. The European Consortium for the study of PHP (EuroPHP) designed the present work with the intention of generating the standards of diagnostic clinical molecular (epi)genetic testing in PHP patients. With this aim, DNA samples of eight independent PHP patients carrying GNAS genetic and/or epigenetic defects (three patients with GNAS deletions, two with 20q uniparental disomy and three with a methylation defect of unknown origin) without GNAS point mutations were anonymized and sent to the five participant laboratories for their routine genetic analysis (methylation-specific (MS)-MLPA, pyrosequencing and EpiTYPER) and interpretations. All laboratories were able to detect methylation defects and, after the data analysis, the Consortium compared the results to define technical advantages and disadvantages of different techniques. To conclude, we propose as first-level investigation in PHP patients copy number and methylation analysis by MS-MLPA. Then, in patients with partial methylation defect, the result should be confirmed by single CpG bisulphite-based methods (ie pyrosequencing), whereas in case of a complete methylation defect without detectable deletion, microsatellites or SNP genotyping should be performed to exclude uniparental disomy 20.

Different pattern of epigenetic changes of the GNAS gene locus in patients with pseudohypoparathyroidism type Ic confirm the heterogeneity of underlying pathomechanisms in this subgroup of pseudohypoparathyroidism and the demand for a new classification of GNAS-related disorders.

CONTEXT: Disorders characterized by PTH resistance are grouped within the term pseudohypoparathyroidism type I (PHPI). Most subtypes of this disease are caused by genetic or epigenetic changes of the GNAS locus leading to deficiency of the α-subunit of stimulatory G proteins (Gsα). Because the in vitro measured Gsα protein activity is normal in pseudohypoparathyroidism Ic (PHPIc), it had previously been postulated that this subtype is caused by impairment of distinct components of the G protein-signaling pathway. However, recently, pathogenic GNAS mutations in a subset of PHPIc patients were found. OBJECTIVE: To clarify the underlying pathogenic mechanism of GNAS exon 1-13 mutation-negative PHPIc cases by investigating the differentially methylated regions of GNAS for epigenetic abnormalities. PATIENTS AND METHODS: The methylation pattern of GNAS exons A/B, AS, XL, and NESP from blood-derived leukocytes of 26 PHPIc patients was assessed by pyrosequencing of bisulfite-converted DNA. RESULTS: Six patients presented with three different patterns of epigenetic changes. One patient had an exclusive loss of methylation of exon A/B associated with a STX16 deletion; four patients had an additional loss of methylation in XL and AS and a gain of methylation in NESP; and one patient presented with partial GNAS methylation changes concerning all differentially methylated regions. CONCLUSIONS: Our results confirm that PHPIc is a heterogeneous entity caused in part by impaired Gsα function, not only due to mutations, but also due to abnormal imprinting of GNAS. However, in the majority of cases of PHPIc, the underlying etiopathogenesis remains elusive.

Defense Mechanisms of Hepatocytes Against Burkholderia pseudomallei.

The Gram-negative facultative intracellular rod Burkholderia pseudomallei causes melioidosis, an infectious disease with a wide range of clinical presentations. Among the observed visceral abscesses, the liver is commonly affected. However, neither this organotropism of B. pseudomallei nor local hepatic defense mechanisms have been thoroughly investigated so far. Own previous studies using electron microscopy of the murine liver after systemic infection of mice indicated that hepatocytes might be capable of killing B. pseudomallei. Therefore, the aim of this study was to further elucidate the interaction of B. pseudomallei with these cells and to analyze the role of hepatocytes in anti-B. pseudomallei host defense. In vitro studies using the human hepatocyte cell line HepG2 revealed that B. pseudomallei can invade these cells. Subsequently, B. pseudomallei is able to escape from the vacuole, to replicate within the cytosol of HepG2 cells involving its type 3 and type 6 secretion systems, and to induce actin tail formation. Furthermore, stimulation of HepG2 cells showed that IFNγ can restrict growth of B. pseudomallei in the early and late phase of infection whereas the combination of IFNγ, IL-1ß, and TNFα is required for the maximal antibacterial activity. This anti-B. pseudomallei defense of HepG2 cells did not seem to be mediated by inducible nitric oxide synthase-derived nitric oxide or NADPH oxidase-derived superoxide. In summary, this is the first study describing B. pseudomallei intracellular life cycle characteristics in hepatocytes and showing that IFNγ-mediated, but nitric oxide- and reactive oxygen species-independent, effector mechanisms are important in anti-B. pseudomallei host defense of hepatocytes.