Rapid DNA Extraction Protocol for Detection of Alpha-1 Antitrypsin Deficiency from Dried Blood Spots by Real-Time PCR

Struniawski, Radosław; Szpechcinski, Adam; Poplawska, Beata; Skronski, Michal; Chorostowska‐Wynimko, Joanna

doi:10.1007/978-94-007-4549-0_5

Cited by 12 publications

(8 citation statements)

References 18 publications

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“…Th e next milestone in the targeted screening program transpired in 2011 when the new method of blood collection for AATD diagnostics was introduced ( 7 ). Th e Dry blood spot (DBS) on fi lter paper technique enabled low-invasive sampling, helped decrease the costs and simplifi ed material shipment.…”

Section: Resultsmentioning

confidence: 99%

The National Alpha-1 Antitrypsin Deficiency Registry in Poland

Chorostowska‐Wynimko¹,

Struniawski²,

Śliwiński³

et al. 2015

COPD: Journal of Chronic Obstructive Pulmonary Disease

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The alpha-1 antitrypsin deficiency (AATD) targeted screening program, together with the National Registry, were established in Poland in 2010 soon after the AATD diagnostics became available. Between 2010 and 2014 a total of 2525 samples were collected from respiratory patients countrywide; 55 patients with severe AAT deficiency or rare mutations were identified and registered, including 36 PiZZ subjects (65%). The majority of AATD patients were diagnosed with COPD (40%) or emphysema (7%), but also with bronchial asthma (16%) and bronchiectasis (13%). Therefore, the registry has proved instrumental in setting-up the AATD-dedicated network of respiratory medical centres in Poland. Since augmentation therapy is not reimbursed in our country, the smoking cessation guidance, optimal pharmacotherapy of respiratory symptoms as well the early detection, and effective treatment of exacerbations is absolutely essential.

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Section: Resultsmentioning

confidence: 99%

The National Alpha-1 Antitrypsin Deficiency Registry in Poland

Chorostowska‐Wynimko¹,

Struniawski²,

Śliwiński³

et al. 2015

COPD: Journal of Chronic Obstructive Pulmonary Disease

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“…The quality and quantity of DNA were checked spectrophotometrically ( NanoDrop One spectrophotometer, Thermo Fisher Scientific, United States). AAT genotyping was performed in the LightCycler 480 II machine (Roche Diagnostics Ltd., Switzerland) using a set of specific oligonucleotide primers and minor groove binding (MGB) hydrolysis probes for PI*S and PI*Z alleles (sequences available upon request; Struniawski et al, 2013 ). Each assay included an S/Z heterozygote, Z/Z homozygote.…”

Section: Methodsmentioning

confidence: 99%

Alpha-1 Antitrypsin Z Variant (AAT PI*Z) as a Risk Factor for Intrahepatic Cholestasis of Pregnancy

et al. 2021

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Background: Intrahepatic cholestasis of pregnancy (ICP; prevalence 0.2–15.6%) is the most common pregnancy-related liver disorder. It may have serious consequences for a pregnancy, including increased risk of preterm delivery, meconium staining of amniotic fluid, fetal bradycardia, distress, and fetal demise. In cases of high bile acids (>100μmol/L), patients have 10-fold increase in the risk of stillbirth. Biophysical methods of fetal monitoring, such as cardiotocography, ultrasonography, or Doppler have been proven unreliable for risk prediction in the course of intrahepatic cholestasis. Therefore, we believe extensive research for more specific, especially early, markers should be carried out. By analogy with cholestasis in children with inherited alpha-1 antitrypsin deficiency (AATD), we hypothesized the SERPINA1 Z pathogenic variant might be related to a higher risk of cholestasis in pregnancy. This study aimed to investigate the most common AATD variants (Z and S SERPINA1 alleles) in a group of cholestatic pregnant women.Results: The Z carrier frequency was calculated to be 6.8%, which is much higher compared to the general population [2.3%; the Chi-squared test with Yates correction is 6.8774 (p=0.008)].Conclusion: Increased prevalence of SERPINA1 PI*Z variant in a group of women with intrahepatic cholestasis may suggest a possible genetic origin of a higher risk of intrahepatic cholestasis in pregnancy.

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“…The nephelometer automatically dilutes analysed samples 1:216 to achieve optimal antigen-antibody equilibrium in the assay. The normal range of the instrument for A1AT in serum samples is 88–174 mg/dL with a cutoff value of 120 mg/dL [ 19 ].…”

Section: Methodsmentioning

confidence: 99%

How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

Ferrarotti

Poplawska-Wisniewska²,

Trevisan

et al. 2015

PLoS ONE

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The Z deficiency in α1-antitrypsin (A1ATD) is an under-recognized condition. Alpha1-antitrypsin (A1AT) is the main protein in the α1-globulin fraction of serum protein electrophoresis (SPE); however, evaluation of the α1-globulin protein fraction has received very little attention. Serum Z-type A1AT manifests in polymeric forms, but their interference with quantitative immunoassays has not been reported. Here, 214 894 samples were evaluated by SPE at the G. Fracastoro Hospital of Verona, Italy. Patients with an A1AT level ≤ 0.92 g/L were recalled to complete A1ATD diagnosis. In parallel, to qualitatively and quantitatively characterize A1AT, sera samples from 10 PiZZ and 10 PiMM subjects obtained at the National Institute of Tuberculosis and Lung Diseases in Warsaw, Poland, were subjected to non-denaturing 7.5% PAGE and 7.5% SDS-PAGE followed by Western blot. Moreover, purified A1AT was heated at 60°C and analyzed by a non-denaturing PAGE and 4–15% gradient SDS-PAGE followed by Western blot as well as by isolelectrofocusing and nephelometry. A total of 966 samples manifested percentages ≤ 2.8 or a double band in the alpha1-zone. According to the nephelometry data, 23 samples were classified as severe (A1AT ≤ 0.49 g/L) and 462 as intermediate (A1AT >0.49≤ 1.0 g/L) A1ATD. Twenty subjects agreed to complete the diagnosis and an additional 21 subjects agreed to family screening. We detected 9 cases with severe and 26 with intermediate A1ATD. Parallel experiments revealed that polymerization of M-type A1AT, when measured by nephelometry or isolelectrofocusing, yields inaccurate results, leading to the erroneous impression that it was Z type and not M-type A1AT. We illustrate the need for confirmation of Z A1AT values by “state of the art” method. Clinicians should consider a more in-depth investigation of A1ATD in patients when they exhibit serum polymers and low α1-globulin protein levels by SPE.

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Rapid DNA Extraction Protocol for Detection of Alpha-1 Antitrypsin Deficiency from Dried Blood Spots by Real-Time PCR

Cited by 12 publications

References 18 publications

The National Alpha-1 Antitrypsin Deficiency Registry in Poland

The National Alpha-1 Antitrypsin Deficiency Registry in Poland

Alpha-1 Antitrypsin Z Variant (AAT PI*Z) as a Risk Factor for Intrahepatic Cholestasis of Pregnancy

How Can We Improve the Detection of Alpha1-Antitrypsin Deficiency?

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