Serum AAT Research Articles

Pathogenic Mutations Conferring α1-Antitrypsin Deficiency Are Identified Accurately by a Novel Multiplexed Molecular Assay

BackgroundAlpha-1 antitrypsin deficiency (AATD) is a common, under-diagnosed disease caused by mutations in the polymorphic SERPINA1 gene. AATD is often causes chronic obstructive pulmonary disease (COPD), other respiratory ailments and/or severe liver disease. AATD under-diagnosis is associated with the lack of a quick, high precision test for SERPINA1 variants. Research QuestionCan a rapid and more accurate molecular diagnostic assay be developed that identifies AATD disease-associated mutations and outperforms current limited methodology? Study Design and MethodsWe developed a multiplexed PCR assay that that uses mass spectrometry to detect 20 pathogenic SERPINA1 mutations, two normal M allele variants and an additional variant of unknown significance, as an accessible frontline genetic test for AATD. Blood samples from 177 patients with AATD indication, 176 presumed normal genotype patients and 10 buccal swabs or blood spots were tested to validate the assay. Additionally, 760 whole blood samples from AATD indication patients were evaluated to identify AATD-associated mutations. ResultsThe novel genotyping assay described here accurately detected 23 SERPINA1 single nucleotide polymorphisms (SNPs) (“AAT SNP23”). Of 177 AATD samples, 96% had abnormal SNPs, whereas 9.1% of the 176 presumed normal samples had abnormal SNPs. AAT SNP23 genotypes correlated well with known serum AAT levels. This genotyping assay was more accurate and streamlined than phenotyping isoelectric focusing assay used to identify AATD variants. For clinical testing, serum AAT protein level determination and AAT SNP23 genotyping assay were performed. AAT SNP23 was successfully implemented using AATD indication patient samples to evaluate the most common SERPINA1 mutations indicative of AATD. AAT SNP23 has allowed for quick and accurate AAT genotyping of patients. InterpretationWe developed a novel, multiplexed genotyping assay that rapidly and accurately identifies multiple AATD-associated SERPINA1 SNPs. This assay is useful to quickly diagnose AATD in patients with pulmonary and/or hepatic diseases of unknown etiology.

Risk of lung disease in the PI*SS genotype of alpha-1 antitrypsin: an EARCO research project

BackgroundThe PI*S variant is one of the most prevalent mutations within alpha-1 antitrypsin deficiency (AATD). The risk of developing AATD-related lung disease in individuals with the PI*SS genotype is poorly defined despite its substantial prevalence. Our study aimed to characterize this genotype and its risk for lung disease and compare it with the PI*ZZ and PI*SZ genotypes using data from the European Alpha-1 antitrypsin Deficiency Research Collaboration international registry.MethodDemographic, clinical, functional, and quality of life (QoL) parameters were assessed to compare the PI*SS characteristics with the PI*SZ and PI*ZZ controls. A propensity score with 1:3 nearest-neighbour matching was performed for the most important confounding variables.ResultsThe study included 1007 individuals, with PI*SS (n = 56; 5.6%), PI*ZZ (n = 578; 57.4%) and PI*SZ (n = 373; 37.0%). The PI*SS population consisted of 58.9% men, with a mean age of 59.2 years and a mean FEV1(% predicted) of 83.4%. Compared to PI*ZZ individuals they had less frequent lung disease (71.4% vs. 82.2%, p = 0.037), COPD (41.4% vs. 60%, p = 0.002), and emphysema (23.2% vs. 51.9%, p < 0.001) and better preserved lung function, fewer exacerbations, lower level of dyspnoea, and better QoL. In contrast, no significant differences were found in the prevalence of lung diseases between PI*SS and PI*SZ, or lung function parameters, exacerbations, dyspnoea, or QoL.ConclusionsWe found that, as expected, the risk of lung disease associated with the PI*SS genotype is significantly lower compared with PI*ZZ, but does not differ from that observed in PI*SZ individuals, despite having higher serum AAT levels.Trial registrationwww.clinicaltrials.gov (ID: NCT04180319).

Transplantace jater pro deficit alfa-1-antitrypsinu

Summary: Alpha-1-antitrypsin deficiency (AATD) is an autosomal codominant genetic condition which manifests with lung emphysema at young age and with liver disease. A point mutation in the SERPINA1 gene, which encodes 1-antitrypsin (AAT), causes a defective secondary structure of the protein, which subsequently accumulates in the endoplasmic reticulum of hepatocytes and is not transported into the blood and body fluids. Deficiency of AAT, which inhibits neutrophil elastase in the lung, thus allows proteolytic damage to the connective tissue of the lung, leading to the development of emphysema. Liver disease is caused by the accumulation of the mutant AAT in liver cells, contributing to proteotoxic liver injury that can lead to liver cirrhosis. Liver transplantation (LT) is the only curative method; after LT, the recipient has the phenotype of the donor organ and normal serum AAT concentrations. The indication for LT should be considered in all patients with AATD and end-stage liver disease. The patients should be referred to the transplant centre when severe complications of liver cirrhosis, such as variceal bleeding, ascites, encephalopathy, and hepatorenal syndrome. In patients with hepatocellular carcinoma, LT represents an optimal treatment modality in the case of an early, unresectable tumour. The timing of LT is crucial; LT should be performed before the patient develops life-threatening complications of end-stage liver disease. LT should be considered in patients of Child-Pugh classification B and C and MELD score of 15 points or higher. Survival of patients after LT for AADT is excellent. Key words: 1-antitrypsin – 1-antitrypsin deficiency – liver cirrhosis – end-stage liver disease – hepatocellular carcinoma – live transplantation

Detection of Alpha-1 Antitrypsin Levels in Chronic Obstructive Pulmonary Disease in Respiratory Clinics in Spain: Results of the EPOCONSUL 2021 Audit.

Alpha-1 antitrypsin deficiency (AATD) is an underdiagnosed condition despite being one of the most common inherited disorders in adults that is associated with an increased risk of developing chronic obstructive pulmonary disease (COPD). The aim was to evaluate the frequency of performing AAT levels and associated factors in COPD patients in an audit conducted in 2021-2022, as well as to compare with a previous audit conducted in 2014-2015. EPOCONSUL 2021 is a cross-sectional audit that evaluated the outpatient care provided to COPD patients in respiratory clinics in Spain based on available data from medical registries. 4225 patients with a diagnosis of COPD from 45 centers were audited in 2021. A total of 1670 (39.5%) patients underwent AAT determination. Being treated at a specialized COPD outpatient clinic (OR 1.88, p = 0.007), age ≤ 55 years old (OR 1.84, p = 0.007) and a FEV1 < 50% (OR 1.86, p < 0.001) were associated with a higher likelihood of being tested for AAT, while Charlson index ≥ 3 (OR 0.63, p < 0.001) and genotyping of AATD availability (OR 0.42, p < 0.001) showed a statistically significant negative association. The analysis of cases included in respiratory units that participated in both audits showed an increase in the proportion of cases with AAT serum level testing available (adjusted OR 2.81, p < 0.001). The percentage of individuals with serum AAT levels < 60 mg/dL (a severe AATD) was 4%. Our analysis identifies significant improvements in adherence to the recommendation to test AAT levels in COPD patients, performed in 4 out of 10 patients, being more likely at younger ages and with higher COPD severity, and with a detection of severe AATD of 4% among those tested, suggesting that clinicians still perform AAT testing in COPD patients selectively. Therefore, efforts are still needed to optimize AATD screening and establish new early detection strategies to reduce morbidity and mortality in these patients.

AAT: does it have a place in acute leukemia?

Aim: In this study, the authors aimed to investigate the change of AAT, its effect on the response to induction and its effects on the treatment process in acute myeloid leukemia and acute lymphoblastic leukemia patients. Materials & methods: This study included 94 patients who were hospitalized and followed up inIstanbul Training and ResearchHospital, Hematology Clinic, between October 2019 and December 2021. Results: Patients with a complete response had higher serum AAT levels than those with a non-complete response (p<0.05). The mean serum AAT level was found to be significantly higher in patients without Gram-positive growth than in patients with Gram-positive growth. Conclusion: It can be thought that AAT can play a role during the course of acute leukemia management.

Prevalence of genetic mutations in alpha-1 antitrypsin deficiency (aatd) in patients with chronic obstructive pulmonary disease in Colombia

BackgroundAlpha-1 antitrypsin deficiency (AATD) is an underrecognized genetic disorder associated mainly with pulmonary emphysema and Chronic Obstructive Pulmonary Disease (COPD). All individuals with COPD regardless of age or ethnicity should be tested for AATD, but in Colombia its prevalence in unknown.Main objectiveTo determine the prevalence of the genetic mutations, present in AATD in adult patients with COPD in Colombia, using a genotyping test on cells from the oral mucosa.MethodsThis was a multicentre, observational, cross-sectional study which included adult patients attending seven COPD care centres in Colombia. Demographic data, medical history, including history of exposure to smoking and biomass smoke, most recent spirometry, pharmacological and non-pharmacological treatment received, serum AAT levels, and mutations detected by the genotyping test were recorded for all the recruited patients. For the comparison of variables between the groups with and without mutation, we used the X2 test for the qualitative variables and the Student’s t-test or Mann-Whitney U test according to their distribution.Main findingsWe collected a sample of 1,107 patients, the median age was 73.8 years (87.6–79.9). Mutations were documented in 144 patients (13.01%), the majority had the M/S mutation (78.50%), followed by M/Z (9.72%). One patient had a ZZ mutation and two patients had null alleles. In total, 23 patients had mutations associated with serum AAT deficiency (levels below 60 mg/dl).ConclusionsGenetic mutations were documented in 13.01% of patients with COPD in Colombia and 2.07% were AATD-related, showing that there is a significant number of underdiagnosed patients.

Normal Alpha-1-Antitrypsin Variants Display in Serum Allele-Specific Protein Levels.

Alpha-1-antitrypsin (A1AT or SERPINA1) has been proposed as a putative biomarker distinguishing healthy from diseased donors throughout several proteomics studies. However, the SERPINA1 gene displays high variability of frequent occurring genotypes among the general population. These different genotypes may affect A1AT expression and serum protein concentrations, and this is often not known, ignored, and/or not reported in serum proteomics studies. Here, we address allele-specific protein serum levels of A1AT in donors carrying the normal M variants of A1AT by measuring the proteoform profiles of purified A1AT from 81 serum samples, originating from 52 donors. When focusing on heterozygous donors, our data clearly reveal a statistically relevant difference in allele-specific protein serum levels of A1AT. In donors with genotype PI*M1VM1A, the experimentally observed ratio was approximately 1:1 (M1V/M1A, 1.00:0.96 ± 0.07, n = 17). For individuals with genotype PI*M1VM2, this ratio was 1:1.28 (M1V/M2, 1.00:1.31, ±0.19, n = 7). For genotypes PI*M1VM3 and PI*M1AM3, a significant higher amount of M3 was observed compared to the M1-subtypes (M1V/M3, 1.00:1.84 ± 0.35, n = 8; M1A/M3, 1.00:1.61 ± 0.33, n = 5). We argue that these observations are important and should be considered when analyzing serum A1AT levels before proposing A1AT as a putative serum biomarker.

Alpha1-antitrypsin deficiency in Greece: Focus on rare variants

PurposeA1Antitrypsin deficiency (AATD) pathogenic mutations are expanding beyond the PI*Z and PI*S to a multitude of rare variants. Aimto investigate genotype and clinical profile of Greeks with AATD. MethodsSymptomatic adult-patients with early-emphysema defined by fixed airway obstruction and computerized-tomography scan and lower than normal serum AAT levels were enrolled from reference centers all over Greece. Samples were analyzed in the AAT Laboratory, University of Marburg-Germany. ResultsIncluded are 45 adults, 38 homozygous or compound heterozygous for pathogenic variants and 7 heterozygous. Homozygous were 57.9% male, 65.8% ever-smokers, median (IQR) age 49.0(42.5–58.5) years, AAT-levels 0.20(0.08–0.26) g/L, FEV1(%predicted) 41.5(28.8–64.5). PI*Z, PI*Q0, and rare deficient allele's frequency was 51.3%, 32.9%,15.8%, respectively. PI*ZZ genotype was 36.8%, PI*Q0Q0 21.1%, PI*MdeficientMdeficient 7.9%, PI*ZQ0 18.4%, PI*Q0Mdeficient 5.3% and PI*Zrare-deficient 10.5%. Genotyping by Luminex detected: p.(Pro393Leu) associated with MHeerlen (M1Ala/M1Val); p.(Leu65Pro) with MProcida; p.(Lys241Ter) with Q0Bellingham; p.(Leu377Phefs*24) with Q0Mattawa (M1Val) and Q0Ourem (M3); p.(Phe76del) with MMalton (M2), MPalermo (M1Val), MNichinan (V) and Q0LaPalma (S); p.(Asp280Val) with PLowell (M1Val); PDuarte (M4), YBarcelona (p.Pro39His). Gene-sequencing (46.7%) detected Q0GraniteFalls, Q0Saint-Etienne, Q0Amersfoort(M1Ala), MWürzburg, NHartfordcity and one novel-variant (c.1A>G) named Q0Attikon.Heterozygous included PI*MQ0Amersfoort(M1Ala), PI*MMProcida, PI*Mp.(Asp280Val), PI*MOFeyzin. AAT-levels were significantly different between genotypes (p = 0.002). ConclusionGenotyping AATD in Greece, a multiplicity of rare variants and a diversity of rare combinations, including unique ones were observed in two thirds of patients, expanding knowledge regarding European geographical trend in rare variants. Gene sequencing was necessary for genetic diagnosis. In the future the detection of rare genotypes may add to personalize preventive and therapeutic measures.

Role and mechanism of alpa1-antitrypsin in polycystic ovary syndrome

To determine the relationships among alpha1-antitrypsin (A1AT), pro-inflammatory cytokines, neutrophil elastase (NE), interleukin (IL)- 1β, and IL-8 in cases with polycystic ovary syndrome (PCOS). Female rats in the control group were fed and watered normally. Female rats in the PCOS group were given high-fat diets and letrozole (1% carboxymethyl cellulose' [CMC] solution) CMC by gavage at a dose of l mg/kg body weight daily for 23 days. The mRNA levels of A1AT and NE in rat ovaries were detected by performing real-time polymerase chain reaction (PCR) in the laboratory of Norman Bethune College of Medicine, Jilin University, China in 2017. All serum samples were collected from the Second Hospital of Jilin University from October 2021 to November 2021 for obesity concurrent with PCOS. Molecular docking of A1AT with NE, IL-8, and IL-1β was investigated using the Insight II software ZDOCK tool. This study was carried out at the Reproductive Center, The Second Hospital of Jilin University, Changchun, China from June 2021 to July 2022. The expression of the A1AT mRNA decreased in the ovary tissues of PCOS rats relative to that of healthy controls, while the expression of NE mRNA increased compared to that of normal controls. The serum A1AT expression in PCOS cases decreased considerably relative to their expression in normal controls. However, the expression of NE, IL-1β, and IL-8 increased significantly relative to their expression in the control (p<0.05 for all). The Insight II ZDOCK molecular docking simulations showed that A1AT has direct interaction sites for NE, IL-1β, and IL-8. Alpha1-antitrypsin is closely associated with NE, IL-1β, and IL-8. Therefore, we speculate that A1AT might ameliorate PCOS symptoms by inhibiting pro-inflammatory factors: NE, IL-1β, and IL-8.

Molecular characterization of PI * S hangzhou , a SERPINA1 allele from continental China encoding a defective alpha-1-antitrypsin.

Alpha-1-antitrypsin deficiency (AATD) is a heritable condition that predisposes to respiratory and hepatic complications. Screenings in East Asia human populations for the AATD alleles most commonly found among Caucasians have yielded poor outcomes. Serum alpha-1-antitrypsin (AAT) levels, AAT phenotypes, and sequences of SERPINA1 gene were examined in a Chinese child with a moderate deficit of serum AAT, who had suffered several episodes of liver disease, as well as in his first-order relatives. Results allowed the identification of PI*Shangzhou, a novel SERPINA1 defective allele, which has been characterized by a L276R substitution, found in a SERPINA1-M3 genetic background. Moreover, potential effects of PI*Shangzhou mutation over the AAT structure were studied by 3D homology modeling. The presence of an arginine residue at position 276 could destabilize the tertiary structure of AAT, since it occurs at a highly conserved hydrophobic cavity in the protein surface, and very close to two positively-charged lysine residues. Attending to the frequency of R276 variant reported in databases for individuals of East Asian ancestry, the PI*Shangzhou allele may explain the global prevalence of the PiS phenotype observed in China.

Frequency of alleles and genotypes associated with alpha-1 antitrypsin deficiency in clinical and general populations: Revelations about underdiagnosis

Background and objectiveAlpha-1 antitrypsin deficiency (AATD) is an underdiagnosed hereditary condition that promotes the development of lung and liver diseases, and the most common potentially life-threatening genetic condition in Caucasian adults. In this study, the clinical and genetic profile of pulmonary patients from a single center in La Palma Island (Canary Islands, Spain) was assessed to predict how to increase AATD diagnosis. MethodsAATD was tested in 1,493 pulmonary outpatients without regard to respiratory symptoms and 465 newborns. Variants of the SERPINA1 gene were characterised by real-time PCR, DNA sequencing, molecular haplotyping and phenotyping (AAT isoelectric focusing). Different respiratory pathologies were diagnosed in patients and their levels of serum AAT were measured by nephelometry. ResultsThe prevalence of pneumological patients with AATD alleles was 30.5%, including PI*S, PI*Z and 6 rare genetic variants. Certain deficiency genotypes were unevenly distributed among patients diagnosed with respiratory diseases: PI*ZZ (71.4%) and PI*SS (34.8%) genotypes were more represented in patients with chronic obstructive pulmonary disease (COPD), whereas PI*MZ (27.7%) and PI*SZ (34.5%) genotypes were more abundant in patients with bronchial asthma. The estimated frequency of PI*S and PI*Z alleles in the general population was 8.2% and 2.1%, respectively. A very significant enrichment (p< 0.01) of PI*S allele, independent of the PI*Z allele, was detected in the clinical population. ConclusionsAATD diagnosis would improve if both the COPD and the asthmatic patients were included to screening programs. The prevalence of PI*ZZ genotype in La Palma (1/2,162) was relatively high within Spain (average 1/3,344).

Maternal alpha-1-antitrypsin as a noval marker for growth restriction in pre-eclampsia.

To verify the role of maternal serum levels of alpha-1-antitrypsin (AAT), an acute-phase inflammatory protein, as a marker for distinguishing between fetal growth restriction (FGR) and normal birth weight in pre-eclamptic women. We correlate serum AAT levels to the essential feto-maternal parameters for an earlier and cost-benefit diagnostic method, thus distinguishing between FGR and normal birth weight in pre-eclamptic women. An observational study conducted at the University hospital recruited 100 pregnant women in 32/34 weeks of a singleton single tone pregnancy; all were pre-eclampsia cases. All were tested by laboratory and ultrasound examination. Two sets of data were collected; one is maternal parameters such as blood pressure (BP), maternal serum AAT mean platelet volume (MPV), platelet distribution width (PDW), and serum uric acid levels, and the other is fetal parameters such as amniotic fluid index (AFI), fetal weight centile and estimated fetal weight. A strong negative correlation proved between serum levels of AAT and all study variables except fetal weight (systolic BP, diastolic BP, MPV, PDW, serum uric acid, fetal weight percentile, and AFI) with a correlation coefficient of; -0.95, -0.95, -0.85, -0.93, -0.91, -0.94, and -0.93 respectively. The cut-off value for AAT 0.013 mg/ml showed the highest sensitivity and specificity as a diagnostic marker for FGR. Area under the curve was 0.99. Negative correlations between maternal serum AAT and fetal parameters used to assess FGR were confirmed, suggesting that AAT is closely related to the pathophysiology of FGR among pre-eclamptic patients and may serve as a helpful tool in distinguishing between FGR and normal birth weight babies, pending further validation in feto-maternal outcomes.

Alpha-1 Antitrypsin Augmentation Inhibits Proteolysis of Neutrophil Membrane Voltage-Gated Proton Channel-1 in Alpha-1 Deficient Individuals.

Background and Objectives: Alpha-1 antitrypsin is a serine protease inhibitor that demonstrates an array of immunomodulatory functions. Individuals with the genetic condition of alpha-1 antitrypsin deficiency (AATD) are at increased risk of early onset emphysematous lung disease. This lung disease is partly driven by neutrophil mediated lung destruction in an environment of low AAT. As peripheral neutrophil hyper-responsiveness in AATD leads to excessive degranulation and increased migration to the airways, we examined the expression of the membrane voltage-gated proton channel-1 (HVCN1), which is integrally linked to neutrophil function. The objectives of this study were to evaluate altered HVCN1 in AATD neutrophils, serine protease-dependent degradation of HVCN1, and to investigate the ability of serum AAT to control HVCN1 expression. Materials and Methods: Circulating neutrophils were purified from AATD patients (n = 20), AATD patients receiving AAT augmentation therapy (n = 3) and healthy controls (n = 20). HVCN1 neutrophil expression was assessed by flow cytometry and Western blot analysis. Neutrophil membrane bound elastase was measured by fluorescence resonance energy transfer. Results: In this study we demonstrated that HVCN1 protein is under-expressed in AATD neutrophils (p = 0.02), suggesting a link between reduced HVCN1 expression and AAT deficiency. We have demonstrated that HVCN1 undergoes significant proteolytic degradation in activated neutrophils (p < 0.0001), primarily due to neutrophil elastase activity (p = 0.0004). In addition, the treatment of AATD individuals with AAT augmentation therapy increased neutrophil plasma membrane HVCN1 expression (p = 0.01). Conclusions: Our results demonstrate reduced levels of HVCN1 in peripheral blood neutrophils that may influence the neutrophil-dominated immune response in the AATD airways and highlights the role of antiprotease treatment and specifically AAT augmentation therapy in protecting neutrophil membrane expression of HVCN1.

Alpha-1 antitrypsin deficiency: clarifying the role of the putative protective threshold.

Alpha-1 antitrypsin deficiency (AATD) is the only readily identifiable monogenic cause of COPD. To date the only condition-specific treatment for AATD-associated COPD is weekly administration of intravenous plasma-purified human alpha-1 antitrypsin (IV-AAT). Uncertainties regarding which AATD genotypes should benefit from IV-AAT persist. IV-AAT is costly and involves weekly administration of a plasma product. Much of the risk stratification has been centred around the long-accepted hypothesis of a "putative protective threshold" of 11 µM (0.57 g·L-1) AAT in serum. This hypothesis has become central to the paradigm of AATD care, although its derivation and accuracy for defining risk of disease remain unclear.We reviewed the literature and examined the association between the 11 µM threshold and clinical outcomes to provide context and insight into the issues surrounding this topic.We found no data demonstrating an increased risk of COPD dependent on the 11 µM threshold. Moreover, an abundance of recent clinical data examining this threshold refutes the hypothesis. Conversely, the use of 11 µM as a treatment target in appropriate ZZ individuals is supported by clinical evidence, although more refined dosing regimens are being explored.Continued use of the 11 µM threshold as a determinant of clinical risk is questionable, perpetuates inappropriate AAT-augmentation practices, may drive increased healthcare expenditure and should not be used as an indicator for commencing treatment.Genotype represents a more proven indicator of risk, with ZZ and rare ZZ-equivalent genotypes independently associated with COPD. New and better risk assessment models are needed to provide individuals diagnosed with AATD with reliable risk estimation and optimised treatment goals.

Alpha-1 Antitrypsin Screening in a Selected Cohort of Patients Affected by Chronic Pulmonary Diseases in Naples, Italy

Introduction. Alpha-1 antitrypsin deficiency (AATD) is a genetic condition associated with several respiratory diseases in patients with severe protein deficiency. AATD is often late diagnosed or underdiagnosed. Diagnosis frequently occurs in patients with chronic obstructive pulmonary disease and emphysema characterized by frequent exacerbations and over ten years’ duration. The purpose of this study was to evaluate the incidence of alpha-1 antitrypsin deficiency in patients with the chronic pulmonary disease after a thorough screening in the city of Naples in southern Italy. Materials and methods. Two hundred patients suffering from respiratory pathology (chronic obstructive pulmonary disease (COPD), emphysema, asthma, or bronchiectasis) were examined and evaluated in our outpatients’ clinic and tested for serum levels of AAT. Patients who had a respiratory disease suspected of AATD and/or serum AAT < 120 mg/dL underwent genetic testing. Genetic screening was performed on samples from 141 patients. Results. A total of 36 patients had an intermediate deficiency of AAT levels. Among them, 8 were PI*MZ, 6 were PI*MS and 22 had rare pathological mutations. Five patients had a severe AATD, all were composite heterozygous with S or Z allele, while the other allele had a rare pathological mutation. Conclusions. The incidence of genetic defects as AATD in the population of patients affected by chronic respiratory disorders is always a matter of discussion because of the frequent interaction between genes and environmental causes. In our series, numerous rare variants and compound heterozygosity have been described. No homozygous patients have been described. The present is one of few studies available on the incidence of rare variants in the geographic area of the city of Naples. So, our results could be considered interesting not only to know the incidence of AATD and its related rare mutations but also to support early diagnosis and treatments for patients with chronic pulmonary disease and frequent exacerbation and to fight the association with environmental causes of pulmonary damages as smoking.

SERPINA1 Gene Promoter Is Differentially Methylated in Peripheral Blood Mononuclear Cells of Pregnant Women

SERine Protein INhibitor-A1 (SERPINA1) is an inducible blood cell gene coding for alpha1-antitrypsin (AAT), a plasma protease inhibitor whose circulating levels are raised during inflammation, infection and advanced pregnancy. DNA methylation has been suggested to play a role in SERPINA1 gene expression regulation in peripheral blood mononuclear cells (PBMCs). The methylation status of SERPINA1 in PBMCs is unknown. The aim of this study was to evaluate the methylation profile of the SERPINA1 promoter in PBMC. To this purpose PBMCs and serum were collected from healthy subjects (HS) (n = 75), including blood donors (BD) (n = 25), pregnant women at early pregnancy (EP) (n = 25), i.e., within the first trimester, and pregnant women at late pregnancy (LP) (n = 25), i.e., at the third trimester. DNA from PBMCs was treated with sodium bisulfite and PCR amplified for SERPINA1 gene promoter, followed by sequencing analyses. AAT serum levels were determined by ELISA test. SERPINA1 was found hypermethylated in 58.7% of HS. The prevalence of SERPINA1 hypermethylation was significantly higher in BD (68%) and EP (88%) than in LP (20%) (p < 0.01). The median serum AAT concentration was 1.07, 0.63, and 3.15 mg/ml in BD, EP, and LP, respectively (p < 0.05, BD and EP vs LP). This study indicates, for the first time, that SERPINA1 gene promoter is differentially methylated in PBMCs from HS. Likely, modulation of the methylation may be a novel epigenetic regulator mechanism of AAT expression in the PBMC of HS. Therefore, SERPINA1 gene promoter methylation may represent an epigenetic biomarker of PBMCs in healthy subjects.

New cis-Acting Variants in PI*S Background Produce Null Phenotypes Causing Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is an inherited condition characterized by reduced levels of serum AAT due to mutations in the SERPINA1 (Serpin family A member 1) gene. The Pi*S (Glu264Val) is one of the most frequent deficient alleles of AATD, showing high incidence in the Iberian Peninsula. Herein, we describe two new alleles carrying an S mutation but producing a null phenotype: QOVigo and QOAachen. The new alleles were identified by sequencing the SERPINA1 gene in three patients who had lower AAT serum levels than expected for the initial genotype. These alleles are the result of combined mutations in cis in a PI*S allele. Sequencing detected the S mutation in cis with Tyr138Cys (S+Tyr138Cys) in two patients, whereas a third one had the S mutation in cis with Pro391Thr variant (S+Pro391Thr). When expressed in a cellular model, these variants caused strong AAT polymerization and very low AAT secretion to almost undetectable levels. The isoelectric focusing method for plasma AAT phenotyping did not show AAT protein encoded by the novel mutant alleles, behaving as null. We called these alleles PI*S-plus because the S variant was phased with another variant conferring more aggressive characteristics to the allele. The current data demonstrate that the clinical variability observed in AATD can be explained by additional genetic variation, such as dual cis-acting variants in the SERPINA1 gene. The possible existence of other unrevealed variants combined in the PI*S alleles should be considered to improve the genetic diagnosis of the patients.

Alpha-1 Antitrypsin Deficiency and Pulmonary Morbidity in Patients with Primary Immunodeficiency Disease: A Single-Center Experience.

Background Alpha-1 antitrypsin deficiency (AATD) is of importance in the pathogenesis of pulmonary emphysema, chronic obstructive pulmonary diseases (COPD), and bronchiectasis. Various pulmonary disorders are a typical feature of primary immunodeficiency disease (PID). This includes recurrent pulmonary infections, immunodysregulation, and autoinflammatory diseases. As a result, incidence of acute and chronic pulmonary diseases is higher. Interestingly, pulmonary morbidity in PID and AATD share similar features. To study the coexistence of AATD in patients suffering from PID, we performed the underlying investigation. Methods We evaluated a study group of 149 patients (n = 149) with PID. In total, serum AAT concentrations were available for 110 patients (n = 110). For the identified patients, we analyzed both clinical associations and interactions. Results Among the investigated patients, reduced serum AAT levels were detected in 7 patients. With regard to the genotype, PI∗ZZ was found in 2 patients, whereas PI∗MZ was observed in 5 patients. Independent of the underlying phenotype, obstructive lung diseases were found in 2 patients with PI∗ZZ and 2 patients with PI∗MZ. Conclusions In Germany, the estimated percentage for PI∗ZZ and PI∗MZ is 0.01% and 1.9%, respectively. As demonstrated, the ratio in our study group was even higher. We identified seven patients with AATD. Since AATD contributes to pulmonary morbidity in PID patients, systematic underdiagnosis of the coexistence might yield a strong clinical impact. Hence, AAT analysis should be offered to all patients with confirmed PID diagnoses. To strengthen this finding, we suggest the investigation of larger databases.

SUN-221 Subclinical Alpha-1 Antitrypsin Deficiency Is Associated with Increased Free Cortisol Fraction in Plasma and Altered Glucocorticoid Delivery to Tissues

BackgroundCorticosteroid Binding Globulin (CBG) binds >85% of plasma cortisol and controls the circulating free cortisol pool. Proteolytic cleavage by neutrophil elastase is proposed to reduce CBG binding affinity and increase free cortisol availability to inflamed tissues. The CORtisol NETwork (CORNET) consortium found that genetic variation at a locus spanning SERPINA1 (encoding alpha-1 antitrypsin, A1AT, the endogenous inhibitor of neutrophil elastase) and SERPINA6 (CBG) contributes to morning total plasma cortisol variation. We hypothesised that A1AT deficiency increases CBG cleavage and hence free plasma cortisol, resulting in increased tissue cortisol delivery in adipose and in HPA axis negative feedback. We tested this in recall-by-genotype studies of people who are heterozygous for inactivating mutations in SERPINA1.Methods16 healthy carriers of one of the two most common A1AT-deficiency single nucleotide polymorphisms (rs17580 & rs28929474) and 16 age-, gender- and BMI-matched controls were recruited from the Generation Scotland Biobank. Participants underwent combined receptor antagonist stimulation of the HPA axis (‘CRASH’) testing using RU486 400mg and spironolactone 200mg, or placebo in a double blind randomised crossover design. Plasma free cortisol was measured by isotopic dilution and ultrafiltration, total cortisol by LC-MS/MS, total CBG by ELISA, CBG binding capacity by radioligand displacement assay, and ACTH by immunoassay. Serum A1AT was measured by ELISA. Tissue cortisol (LC-MS/MS) and expression of glucocorticoid dependent transcripts (qPCR) were measured in subcutaneous adipose samples collected by needle biopsy.ResultsSerum A1AT was confirmed lower in those with heterozygous mutations vs wild type controls (411.3 +/- 27.44 vs 565.1 +/- 23.38 mg/dL, p=0.0002). No measurable differences in total CBG or CBG binding capacity were observed. However, plasma free cortisol fraction was higher in those carrying A1AT mutations (16.13 +/- 0.2 vs 13.88 +/- 0.04 %, p<0.0001). Adipose cortisol concentrations were not significantly different but expression of glucocorticoid responsive genes e.g. PER1 was 54% higher (p=0.014) in A1AT-deficient subjects. Plasma cortisol was elevated during CRASH testing in both groups, with the increment versus placebo tending to be lower in A1AT-deficient subjects (82.5 +/- 6.7 vs 126.7 +/- 6.8 nM).ConclusionAlpha-1 antitrypsin mutation heterozygosity, common in the general population, is associated with higher free cortisol fraction, consistent with enhanced cleavage of CBG. This is associated with evidence of enhanced delivery of glucocorticoid to adipose tissues but reduced HPA negative feedback, suggesting tissue-specific control of cortisol delivery by CBG.

Alpha-1-antitrypsin molecular testing in Canada: A seven year, multi-centre comparison

BackgroundLaboratory confirmation of alpha-1-antitrypsin (A1AT) deficiency may be achieved by multiple methods. Here, we compare the relative comprehensiveness and efficiency of pathogenic variant (PV) detection of four different protocols utilized at different diagnostic centres in Canada. MethodsDiagnostic results from 2011 to 2018 at clinical laboratories in British Columbia (BC), Alberta (AB), Ontario (ON), and Québec (QC) were reviewed. The four labs utilize the following protocols: BC-CGID (serum A1AT Concentration/Genotyping/Isoelectric focussing (IEF)/SERPINA1 DNA sequencing), AB-CID (serum A1AT Concentration/IEF/DNA sequencing), ON-CD (serum A1AT Concentration/DNA sequencing), and QC-G (Genotyping). As the respective catchment areas varied in size and ethnic composition, the comprehensiveness of PV detection was assessed by comparing the frequency of individual genotypes to the ZZ genotype, which is clearly identified by all protocols. ResultsCollectively 5399 index patients were tested identifying 396 ZZ genotypes. Serum A1AT concentration as a determinant of further testing efficiently identified PV. ON-CD had the highest detection rate for PV; genotypes with at least one PV, other than S, Z or F, were identified at 0.67/ZZ as compared to <0.2/ZZ (all others). However, ON-CD had the highest rates of undefined molecular variants (UMV) (0.16/ZZ) or likely benign variants (LBV) (0.08/ZZ), compared to all others (<0.12/ZZ and < 0.06/ZZ, respectively). The F variant was identified at 0.10/ZZ, only in the ON-CD and the AB-CID protocols. Collectively, MMalton was the next most common variant, identified as a compound heterozygous genotype at 0.04/ZZ, only in the ON-CD and BC-CGID protocols. ConclusionStrategies which readily detect variants across the full coding sequence of SERPINA1 detect more PV as well as more UMV and LBV.