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Summary: Targeted detection programmes are recommended to identify subjects affected by severe alpha1-antitrypsin deficiency (AATD). Guidelines are available to address physicians towards subjects at high risk for AATD. We wanted to investigate the clinical characteristics of subjects enrolled in the programme, who result as not being affected by severe AATD; this information is not available in the present literature. We elaborated data contained in the questionnaires accompanying the samples of 2127 Italian subjects submitted for AATD detection in a period spanning 11 years (1996–2006). A total of 588 subjects were eligible to enter this study: PI*MM subjects and subjects with intermediate AATD, referred for lung disease, were characterised by a relatively young mean age, and a high proportion (31.2% and 28.6%, respectively) were never smokers. Fifty percent or more had symptoms of chronic bronchitis, but without obstruction. Only a minority belonged to most severe GOLD stages. The mean levels of AAT varied as a function of the presence or absence of airflow obstruction in intermediate AATD subjects, but not in PI*MM. Individuals enrolled in AATD detection programmes represent an interesting cohort both for public health and research purposes. [Copyright &y& Elsevier]

The laboratory diagnosis of alpha(1)-antitrypsin (AAT) deficiency (AATD) has evolved over the last 40 years since the first cases of the disorder were reported. It is currently performed in specialized centers, and it requires a combination of different biochemical methods: nephelometric AAT concentration, isoelectric focusing, genotyping, and sequencing. The availability of matrices such as the dried blood spot have facilitated the implementation of laboratory analyses for AATD, but they have also challenged laboratories to develop more reliable and reproducible techniques starting from dried blood. In this article, we describe the protocols we have optimized for AATD diagnosis from dried blood spot, in an attempt to hopefully provide useful information for physicians and scientists involved in this diagnostic line. We also describe the diagnostic flowchart for AATD detection that we have developed accordingly. [ABSTRACT FROM AUTHOR]

Rationale: The receptor for advanced glycation end products (RAGE) engages a number of ligands implicated in inflammatory processes. The RAGE coding gene maps to the 6p21.32 region, close to the genes DRB1 and BTNL2, which are associated with sarcoidosis. Objectives: We investigated a possible implication of RAGE in sarcoid granulomas. Methods: RAGE and major ligands (N-epsilon-carboxy-methyl-lysine [CML], S100A12, and S100B) expression was investigated by immunostaining of 99 paraffin-embedded biopsies of sarcoid tissues, and expression patterns were determined. Among the three RAGE gene single-nucleotide polymorphisms investigated, -374 T/A was selected, characterized in terms of transcriptional effect (immunocytochemistry and real-time polymerase chain reaction), and its frequency was determined in DNA extracted from biopsies. Measurements and Results: RAGE, CML, S100A12, and S100B immunoreactivity was observed in all sarcoid granulomas, although at different intensities. The degree of RAGE expression significantly correlated with the degree of S100A12 expression. The -374 TT/AT genotypes, associated with higher RAGE transcriptional activity, were more frequent in the sarcoidosis biopsy group than in control subjects, and the association was confirmed in a second, independent series of 101 patients with sarcoidosis. Conclusions: We showed the association of RAGE and its ligands with sarcoidosis and suggest that an intrinsic genetic factor could be in part involved in its expression. In Italian patients, the -374 T/A polymorphism seems to be significantly associated with this disease. [ABSTRACT FROM AUTHOR]

Abstract Background AATD is one of the most common inherited disorders in the World. However, it is generally accepted that AATD in North African populations is not a risk factor for lung and/or liver disease, based on a number of small studies. We therefore planned a screening study for detection of AATD in patients with OLD in a cohort of patients from Kairouan in central Tunisia. Methods: One hundred twenty patients with OLD (asthma, emphysema, COPD) were enrolled in the screening programme. Laboratory diagnosis for AATD was performed according to current diagnostic standards. Results We found that 6/120 OLD patients carried an AAT deficient allele, 1 PI*MZ, 1 PI*MPlowel, 3 PI*MMmalton, 1 PI*MMwurzburg. Conclusion this pilot study demonstrated that alleles related to deficiency of AAT are not absent in the Tunisian population, and that rare AATD variants prevailed over commonest PI*Z variant. These results would support a larger scale screening for AATD in Tunisia.

Purpose: Knowledge of tumor mutational status has become a priority for effective NSCLC-tailored treatment. NSCLC diagnosis is more often reached through biopsy; thus, there is a clear need to implement for routine tumor molecular profiling on small cytological samples. This work aims to screen and compare the EGFR and KRAS mutational prevalence in fresh tumor cells and in corresponding routinely processed samples derived from trans-thoracic fine-needle aspiration. The latter currently represents the most appropriate diagnostic procedure in case of peripheral lesions, such as adenocarcinomas, which account for almost 40% of all NSCLCs and for the highest EGFR mutational rates., Methods: Two hundred and forty-four patients carrying peripheral lung masses underwent CT-guided aspiration. The obtained material was split, and a part was addressed to conventional histopathological analysis while the remaining one was stored at -20 °C. In case of confirmation of adenocarcinoma, tumor genomic DNA was extracted from both fresh and fixed material, and EGFR and KRAS sequencing was performed., Results: We identified 136 adenocarcinomas; from 134, we could recover enough material for the study. A full match was demonstrated between EGFR/KRAS mutational prevalences through the two approaches tested. We found EGFR mutations in 13 patients (9.7%); 7 were females and 11 never or former smokers. KRAS mutations occurred in 20 (14.9%) patients. EGFR and KRAS mutations were mutually exclusive., Conclusions: Mutational screening on fresh cancer cells is an achievable, safe and cost-effective procedure which might allow routinely tumor molecular profiling as powerful integration of conventional histopathological analysis.

Cancer is a genetic disease and this concept is now widely exploited by both scientists and clinicians to design new targeted molecules. Indeed many data have already allowed us to ameliorate not only our knowledge about cancer onset, but also about patients treatment. Correlation between mutations in cancer alleles and drug response is a key point to identify drugs that match the genetic profile of each individual tumors. On the other hand, experience derived from inhibition of tyrosine kinase receptors has pointed out that targeted treatment is really successful only in a small subset of tumors. The latter are eventually addicted to those genetic alterations which are responsible for receptors activation and for the continued expression of their signalling. Overall these observations provide a strong rationale for a molecular-based diagnosis and patients selection for targeted therapies. This review analyses the current state of the art of molecularly-tailored pharmacological approach to lung cancer, one of the biggest killers among human solid tumors. Main relevance is addressed to genetic lesions activating the EGFR pathway transducers, focusing on their role as markers of targeted drug response., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Alpha1-antitrypsin (AAT) deficiency is a hereditary disorder associated with reduced AAT plasma levels, predisposing adults to pulmonary emphysema. The most common genetic AAT variants found in patients are the mildly deficient S and the severely deficient Z alleles, but several other pathogenic rare alleles have been reported. While the plasma AAT deficiency is a common trait of the disease, only a few AAT variants, including the prototypic Z AAT and some rare variants, form cytotoxic polymers in the endoplasmic reticulum of hepatocytes and predispose to liver disease. Here we report the identification of three new rare AAT variants associated to reduced plasma levels and characterize their molecular behaviour in cellular models. The variants, called Mpisa (Lys259Ile), Etaurisano (Lys368Glu) and Yorzinuovi (Pro391His), showed reduced secretion compared to control M AAT, and accumulated to different extents in the cells as ordered polymeric structures resembling those formed by the Z variant. Structural analysis of the mutations showed that they may facilitate polymerization both by loosening 'latch' interactions constraining the AAT reactive loop and through effects on core packing. In conclusion, the new AAT deficiency variants, besides increasing the risk of lung disease, may predispose to liver disease, particularly if associated with the common Z variant. The new mutations cluster structurally, thus defining a region of the AAT molecule critical for regulating its conformational state.

The first step in laboratory diagnosis of alpha1-antitrypsin deficiency (AATD) is the determination of alpha1-antitrypsin (AAT) serum levels; these levels in turn are influenced by the inflammatory status. C reactive protein (CRP) has been proposed as a marker of systemic inflammation. Single nucleotide polymorphisms (SNPs) in the CRP gene have been associated with differences in baseline CRP levels. The purpose of this study was to investigate the relationship between CRP and AAT in the AATD diagnostic setting and to verify whether variations in the CRP gene could influence CRP. We determined AAT and CRP levels in 362 consecutive dried blood spot (DBS) samples submitted for AATD diagnosis and genotyped 3 CRP gene SNPs (rs1205, rs3093077, and rs3091244) associated with variations in serum CRP concentrations. To this aim, we developed a method to measure CRP in a DBS with a good correlation with CRP measurement in serum (r2=0.9927). We showed then that systemic inflammatory status parallels increased levels of AAT (80% of subjects with intermediate AATD and a CRP>0.8 mg/dL had an AAT level above the cut-off of 113 mg/dL) and that this increase might mask the presence of AATD variants. No association was detected between CRP levels and the 3 CRP gene polymorphisms. Simultaneous determination of CRP and AAT is useful in the correct diagnosis of heterozygotes carrying intermediate AATD genotypes; their genetic influence on the CRP level is negligible., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Clinical trials to evaluate patients affected by rare diseases are often hampered by the difficulty of recruiting a critical sample size. Registries for rare conditions are thus extremely powerful tools for overcoming recruitment problems. Here we present and discuss the international experience with alpha1-antitrypsin deficiency achieved by the Alpha One International Registry, and national experience obtained with a large series of patients with pulmonary alveolar proteinosis., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Alpha1-antitrypsin (alpha(1)AT) deficiency is a hereditary disorder associated with reduced alpha(1)AT serum level, predisposing adults to pulmonary emphysema. Among the known mutations of the alpha(1)AT gene (SERPINA1) causing alpha(1)AT deficiency, a few alleles, particularly the Z allele, may also predispose adults to liver disease. We have characterized a new defective alpha(1)AT allele (c.745G>C) coding for a mutant alpha(1)AT (Gly225Arg), named P(brescia). The P(brescia) alpha(1)AT allele was first identified in combination with the rare defective M(würzburg) allele in an 11-year-old boy showing significantly reduced serum alpha(1)AT level. Subsequently, the P(brescia) allele was found in the heterozygous state with the normal M or the defective Z allele in nine and three adults respectively. In cellular models of the disease, we show that the P(brescia) mutant is retained in the endoplasmic reticulum as ordered polymers and is secreted more slowly than the normal M alpha(1)AT. This behaviour recapitulates the abnormal cellular handling and fate of the Z alpha(1)AT and suggests that the mutation present in the P(brescia) alpha(1)AT causes a conformational change of the protein which, by favouring polymer formation, is etiologic to both severe alpha(1)AT deficiency in the plasma and toxic protein-overload in the liver.

Background: AATD is one of the most common inherited disorders in the World. However, it is generally accepted that AATD in North African populations is not a risk factor for lung and/or liver disease, based on a number of small studies. We therefore planned a screening study for detection of AATD in patients with OLD in a cohort of patients from Kairouan in central Tunisia., Methods: One hundred twenty patients with OLD (asthma, emphysema, COPD) were enrolled in the screening programme. Laboratory diagnosis for AATD was performed according to current diagnostic standards., Results: We found that 6/120 OLD patients carried an AAT deficient allele, 1 PI*MZ, 1 PI*MPlowel, 3 PI*MMmalton, 1 PI*MMwurzburg., Conclusion: this pilot study demonstrated that alleles related to deficiency of AAT are not absent in the Tunisian population, and that rare AATD variants prevailed over commonest PI*Z variant. These results would support a larger scale screening for AATD in Tunisia.

Background: Individuals with severe deficiency in serum alpha(1)-antitrypsin (AAT) concentrations are at high risk for developing chronic obstructive pulmonary disease (COPD), whereas those carrying the PI*MZ genotype are at slightly increased risk. Testing appropriate subgroups of the population for AAT deficiency (AATD) is therefore an important aspect of COPD prevention and timely treatment. We decided to perform an exhaustive investigation of SERPINA1 gene variants in individuals from the general population with a moderately reduced serum AAT concentration, because such information is currently unavailable., Methods: We determined the Z and S alleles of 1399 individuals enrolled in the Swiss Cohort Study on Air Pollution and Lung Diseases in Adults (SAPALDIA) with serum AAT concentrations < or = 1.13 g/L and submitted 423 of these samples for complete exon 2-->5 sequencing., Results: We found that 900 of 1399 samples (64%), carried the normal PI*MM genotype, whereas 499 samples (36%) carried at least 1 SERPINA1 deficiency variant. In the subpopulations in which AAT concentrations ranged from > 1.03 to < or = 1.13 and from > 0.93 to < or = 1.03 g/L, individuals with the PI*MM genotype represented the majority (86.5% and 53.8%, respectively). The PI*MS genotype was predominant (54.9%) in the AAT range of 0.83 to 0.93 g/L, whereas PI*MZ represented 76.4% in the AAT range of > 0.73 to < or = 0.83 g/L., Conclusions: This analysis provided a detailed molecular definition of intermediate AATD, which would be helpful in the diagnostic setting.

Background: Micro-organisms, behaving in a non-infectious fashion, may be among the exogenous factor(s) believed to trigger idiopathic pulmonary fibrosis (IPF). One possible strategy to identify an individual's susceptibility to such microbial triggers, which are likely to be ubiquitous, is to investigate the molecular processes involved in their recognition. NOD2/CARD15 is a specific pattern recognition receptor protein, whose genetic variants have been previously associated with susceptibility to Crohn's disease., Aim: The aim of this work was to determine the frequencies of the three major NOD2/CARD 15 gene mutations (R702W, G908R and 1007fsinC) in a series of 76 subjects affected by IPF, and to compare them with those found in three groups of controls: a group with sarcoidosis (a disorder in which an involvement of the NOD2/CARD15 gene has already been investigated and rejected in different ethnic groups; 67 subjects) and two groups of healthy subjects (218 and 208 subjects, respectively), matched for gender, age, and ethnicity., Results: We found no differences in frequencies of NOD2/CARD15 gene polymorphisms among the four groups investigated., Conclusion: We conclude that the NOD2/CARD15 gene is not likely to be involved in susceptibility to IPF in Italians.

Alpha1-antitrypsin deficiency (AATD) is a common hereditary disorder associated with high risk of developing pulmonary emphysema early in life and, to a lesser extent, chronic liver disease and cirrhosis. Among Northern Europeans and Northern Americans, more than 95% of individuals with emphysema associated with AATD carry the most frequent AAT deficient gene variants, PI*Z and PI*S. Rare AAT deficient variants account for 2-4% of AATD individuals. We extend the sequence data on AAT by characterizing a novel Null allele detected in 3 subjects: a carrier belonging to an Italian/Egyptian family and 2 members of a family originating from Southern Italy. The mutation raised on a M1 (Ala213) base allele and it is characterized by an A-->T transversion at exon III, nt 218, codon 259 (AAA-->TAA) (GeneBank accession number AY 256958). The transversion results in a premature stop codon (Lys259AAA-->Stop259TAA). The proposed nomenclature of Q0cairo is from the birthplace of the father of first recognized subject. Serum levels and isoelectric focusing of AAT were consistent with the presence of the Null variant.

There is worldwide growing awareness of alpha 1-antitrypsin deficiency (AATD), a major hereditary disorder in Caucasians. The gold standard for laboratory diagnosis of AATD is thin-layer isoelectrofocusing (IEF), which is labor intensive and should be performed in reference laboratories. The aim of this study was to find an easy, fast, and cheap method for detecting alpha1-antitrypsin S and Z variants, the most frequent variants associated with AATD. The novel method herein described is based on SexAI/Hpy99I RFLP. We studied samples from 90 subjects enrolled in the Italian National Registry for AATD, previously typed by isoelectrofocusing. We found a complete agreement among our results, IEF, and genotypes obtained by standard methods. We concluded that this novel method combines efficiency, ease, swiftness, and low cost.

Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrotic disorder underlain by aberrant wound healing of repeated lung injury. Environmental triggers and genetic background are likely to act as modifiers of the fibrotic response. Erythrocyte complement receptor 1 is a membrane protein mediating the transport of immune complexes to phagocytes. Three gene polymorphisms are related to the erythrocyte surface density of complement receptor 1 molecules, which in turn are related to the rate of immune complexes' clearance. There is evidence of association between sarcoidosis and the complement receptor 1 gene. We wondered whether IPF is associated with the complement receptor 1 gene alleles coding for a reduced molecule/erythrocyte ratio. We studied 74 patients and 166 control subjects. Three polymorphic sites of the gene, A3650G exon 22, HindIII RFLP intron 27, and C5507G exon 33, were analyzed and found to be in linkage disequilibrium. The GG genotype for the C5507G exon 33 polymorphism was significantly more common in patients with IPF than in control subjects (odds ratio = 6.232, 95% confidence interval = 2.198-18.419, p = 0.00023). The significant difference was found in both sexes. These findings agree with speculations on the role of the complement receptor 1 gene in idiopathic pulmonary fibrosis.