Alessandro Marocchi

Pru p 3-Sensitised Italian Peach-Allergic Patients Are Less Likely to Develop Severe Symptoms When Also Presenting IgE Antibodies to Pru p 1 and Pru p 4

Background: The roles played by different peach allergens with respect to symptom severity have not been completely ascertained. We have evaluated the diagnostic efficacy of peach recombinant allergens ImmunoCAP compared to peach in the identification of subjects at an increased risk for severe reactions to peaches. Methods: 148 peach-allergic patients were divided based on their symptom severity into 2 groups: mild oral allergy syndrome (OAS) and severe OAS. Anti-rPru p 1, 3 and 4 IgE levels were measured. Statistical analyses were carried out using parametric and non-parametric tests. Results: anti-rPru p 1 and anti-rPru p 4 IgE levels were significantly higher in patients with mild OAS than in patients with severe OAS (p = 0.0001); in contrast, anti-rPru p 3 IgE levels were significantly higher in patients with severe OAS than in patients with mild OAS (p < 0.00005). Moreover, we found that any unitary increase in anti-rPru p 1 IgE values corresponded to a 2.48% reduction in the odds of having severe OAS (p = 0.048), whereas any unitary increase in anti-rPru p 3 IgE values corresponded to a 9.02% increase in the probability of having severe OAS (p = 0.001). Unexpectedly, we found that patients positive to rPru p 3 as well as rPru p 1 and 4 demonstrated a significant reduction of the odds of developing severe symptoms than those positive to rPru p 3 alone. Anti-rPru p 3 IgE levels were a significantly better indicator than anti-peach IgE values (p = 0.016) of patients with the highest risk for severe OAS. A cutoff of 2.69 kUA/l for anti-rPru p 3 IgE values better discriminated peach-allergic patients at a higher risk for symptoms. Conclusions: Italian patients with positive anti-rPru p 1, 4 and 3 IgE levels seemed less likely to experience the clinical effects of high anti-rPru p 3 IgE values.

Frequency of butyrylcholinesterase gene mutations in individuals with abnormal inhibition numbers: an Italian-population study.

OBJECTIVES More than 30 genetic variants of serum cholinesterase (butyrylcholinesterase, BChE) have been described. Some of them (the atypical and the fluoride-resistant variants) are well known because carriers are prone to develop prolonged apnea following the administration of the muscle relaxant succinylcholine. Genotype characterization is therefore important in order to prevent such episodes. Genetic studies have so far focused on selected individuals or families rather than on the random population. METHODS From a large group of healthy blood donors (n = 2609), we selected all the 58 individuals with low serum cholinesterase activity: among them 28 subjects had abnormal dibucaine and fluoride inhibition numbers. Twenty-five mutations in the coding region of the human cholinesterase gene were analyzed. RESULTS All individuals with abnormal inhibition numbers were homozygotes or double heterozygotes in several mutations. Asp70Gly (Atypical variant) and Ala539Thr (K variant) were the most frequently observed amino acid substitutions. The majority of subjects with low BChE activity but normal dibucaine and fluoride number presented only the K form. We analyzed 106 randomly chosen subjects for K and atypical variants. Carriers of these alleles were at risk of low BChE activity (OR = 9.55, 95%CI, 5.61-16.26 and OR = 30.33, 95%CI, 7.05-130.52 respectively). CONCLUSIONS Data obtained from this study help to better define the etiology of low BChE activity and the role of the rather common K allele. It is the first time that such a large population has been screened for so many mutations. BChE is also implicated in detoxifying cocaine; therefore genetic analysis could be useful in cases of cocaine toxicity in Italian subjects.

Phenotypic Heterogeneity in a SOD1 G93D Italian ALS Family: An Example of Human Model to Study a Complex Disease

We report different clinical expression in seven members of a large family with amyotrophic lateral sclerosis (ALS) and the G93D mutation in exon 4 of the Cu/Zn superoxide dismutase (SOD1) gene. The ALS clinical course in the proband showed an unusually fast progression of the disease compared to the paucisymptomatic presentation associated to this mutation in the two previously Italian families described. The remaining mutation carriers did not show the aggressive clinical course displayed by the proband. We selected few genes known to be ALS modifiers searching for genetic variants that could explain the wide phenotypic diversity within the family. Exclusion of causative genes such as TDP43, FUS, PGRN and VAPB was performed too. We believe that this kind of family with contrasting phenotypes of ALS may be considered an excellent human model to study the relationship between a wider genetic profile, including modifier genes, and the clinical expression of the disease. Therefore, the novelty of our approach is also represented by the study of a single family to reproduce a composite structure in which search for possible modifier genes/genetic variants linked to SOD1 mutated.

Mutation in the senataxin gene found in a patient affected by familial ALS with juvenile onset and slow progression

Abstract We report an Italian male with juvenile onset familial disease characterized by progressive weakness and wasting of four limbs and prolonged survival. Diagnostic work-up revealed the diffuse involvement of central and peripheral motor neurons. Genetic analysis revealed a L389S mutation in the senataxin (SETX) gene.

Genetic variability of the fructosamine 3-kinase gene in diabetic patients.

Abstract Background: Nonenzymatic glycation appears to be an important factor in the pathogenesis of diabetic complications. Fructosamine 3-kinase (FN3K), initially identified in erythrocytes, appears to be responsible for the removal of fructosamine from proteins, suggesting a protective role in nonenzymatic glycation. Recently, genetic variants in the FN3K gene have been studied in diabetic patients. The aim of our study was the molecular characterization of the FN3K gene in a representative group of Italian patients with type 1 (T1DM) and 2 (T2DM) diabetes mellitus and in a cohort of healthy controls. Methods: Seventy diabetic subjects (35 type 1 and 35 type 2) with stable glycemic control and 33 healthy control subjects were evaluated using PCR and direct sequencing of the FN3K gene. Denaturing high performance liquid chromatography (DHPLC) was used in controls for screening for the presence of the genetic variants previously found in diabetic patients. Results: Seven different genetic variants were identified, five of them already reported and two new: the p.R187X and p.Y239C mutations identified in two females affected by T2DM. No significant association was found between certain polymorphisms and diabetes conditions. Preliminary haplotype studies are also reported. With respect to genotypes, we noted that some were not present in all the investigated cohort, and some were found related to higher glycated hemoglobin compared to others, although not at a significant level, probably because of the small number of subjects investigated. Conclusions: In conclusion, this study identified two new mutations and additional variants within the FN3K gene. This is the first study on FN3K in Italy. Future work is needed to achieve a better understanding of the FN3K enzyme and its possible clinical utility in the management of diabetic patients.

Molecular screening test in familial forms of cerebral cavernous malformation: the impact of the Multiplex Ligation-dependent Probe Amplification approach

Object The purpose of this study was to underline the effectiveness of molecular analysis in cerebral cavernous angioma, with special attention to the familial forms. Methods Multiplex Ligation-dependent Probe Amplification analysis integrates the consecutive sequence analysis of the 3 genes (Krit1/CCM1, MGC4607/CCM2, and PDCD10/CCM3) known to be responsible for cerebral cavernous malformation lesions. Results The Multiplex Ligation-dependent Probe Amplification analysis revealed a new mutation, a heterozygous exon 9/10 deletion of Krit1, in the proband and in all affected family members. Conclusions The identification of the molecular defect allows physicians to screen family members at risk and to identify affected individuals before the onset of clinical symptoms caused by the presence of lesions.

Total serum tryptase: a predictive marker for KIT mutation in acute myeloid leukemia.

Human tryptase is a serine protease expressed in mast-cells. We previously observed that AML blast cells, cultured in vitro from a KIT D816Y patient, give rise to adherent cells with mast-cell like phenotype and tryptase was released in the serum-free medium. To correlate total serum tryptase (ts-try) levels with cytogenetic features and KIT mutational status, we analyzed serum samples from AML patients at diagnosis. In 70 out of 155 patients (45%) we detected elevated ts-try (>15 ng/mL), significantly linked to t(8;21) (P < .001) and inv(16) (P = .007). In patients that achieved complete remission the ts-try decreased to normal values. In 75 patients screened for KIT mutation, we found a clear relationship between elevated ts-try and mutated patients with t(8;21) (P < .001). In conclusion, we propose that checking for ts-try at diagnosis of AML may be a simple tool to select patients to be addressed to KIT mutation screening.

A two-center evaluation of the blood gas Immediate Response Mobile Analyzer (IRMA)

Abstract The Immediate Response Mobile Analyzer (IRMA) is a selective and portable point-of-care testing (POCT) blood gas, electrolyte and hematocrit (Hct) analyzer. The overall analytical performance was evaluated in a two-center study involving two Italian hospital laboratories, following the guidelines suggested by the manufacturer (based on the NCCLS protocol), after a preliminary evaluation of their formal validity. The IRMA was compared to the analyzers used in the routine laboratory as reference. The considered parameters were pH, pO2, pCO2, Na+, K+, ionized calcium and Hct. When using the aqueous quality control material provided by the manufacturer most of the parameters showed good precision, with the exception of pCO2 and pO2 that showed high CVs on two of the three levels of the aqueous control. We could demonstrate that this imprecision was material-related and was reduced when using a different material (blood equilibrated by tonometry). With tonometred blood for pO2 and pCO2 and the aqueous material for the remaining parameters the CVs were all below 5%, ranging from 0.08% to 2.8%. The IRMA results correlated adequately with the comparison instruments, with the exception of sodium and ionized calcium where contradictory results were obtained in the two centers.

Genetic Variations Within KRIT1/CCM1, MGC4607/CCM2 and PDCD10/CCM3 in a Large Italian Family Harbouring a Krit1/CCM1 Mutation

Cerebral cavernous malformations (CCMs) are congenital vascular anomalies of the central nervous system that can result in seizures, haemorrhage, recurrent headaches and focal neurologic deficit. CCMs can occur as an autosomal dominant trait with incomplete penetrance and a wide phenotypic variability. The genes responsible for this disease are KRIT1/CCM1 on chromosome 7q21.2, MGC4607/CCM2 on chromosome 7p15–p13 and PDCD10/CCM3 on chromosome 3q25.2–q27. Mutations in KRIT1/CCM1 account for more than 40% of CCMs. We previously reported a CCM family harbouring the KRIT1/CCM1 1204delAACAA mutation. In order to search for possible explanation of the clinical variability observed, we looked for genetic variation within exons and exon/intron regions in the three genes KRIT1, MGC4607 and PDCD10 associated to the disease within this large family, 23 subjects have been analysed. Identified genetic variations in the three genes are here presented. We believe that genetic variations could interfere with the proper CCM1/CCM2/CCM3 protein complex thus explaining the observed clinical variability.

De Novo MGC4607 Gene Heterozygous Missense Variants in a Child with Multiple Cerebral Cavernous Malformations

Cavernous malformations are angiographically occult, low-pressure neurovascular lesions with distinct imaging and clinical characteristics; main clinical manifestations are seizure, focal neurological deficits and epileptic attacks. Here we describe the molecular characterization of an Italian child, a symptomatic patient, affected by multiple cerebral cavernous malformations, without a family history of the disease and harbouring a new MGC4607 gene mutation. We identified two de novo missense variants in exon 6 of the gene both present on the same allele (cis configuration). DNA analysis for KRIT1, and PDCD10 gene variation through direct sequencing and MLPA analysis excluded further mutations. STR multiplex assay, allele-specific analysis and DHPLC analysis were performed for a better genetic characterization. Our findings emphasize the importance of the genetic test in subjects presenting multiple cerebral cavernomas for an adequate counselling, as well as for disease management since early identification of genetic abnormalities enable patients to have their lesions removed before they haemorrhage and cause deficit and/or epilepsy.