Rapid detection of Mmalton α1-antitrypsin deficiency allele by real-time PCR and melting curves in whole blood, serum and dried blood spot samples

Belmonte, Irene; Montoto, Luciana; Miravitlles, Marc; Barrecheguren, Miriam; Esquinas, Cristina; Rodrı́guez, Esther; Giralt, Marina; Rodríguez‐Frías, Francisco

doi:10.1515/cclm-2015-0297

Cited by 11 publications

(9 citation statements)

References 19 publications

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“…The protocol for Mmalton allele-specific genotyping has been described in detail elsewhere 17. In cases in which the Mmalton allele was not found, direct sequencing was performed after PCR amplification of all the encoding exons (II–V) using a whole blood sample.…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, since Mmalton and other rare variants are not easily recognizable, this may contribute to misclassification of many of these cases, with the subsequent underestimation of their true frequency 16. For this reason, in our laboratory, we have recently developed a rapid real-time polymerase chain reaction (PCR) and melting curves assay designed for the detection of the Mmalton allele 17. This method detects the Mmalton mutation by analysis of the melting point of one of the two adjacent fluorescently labeled probes.…”

Section: Introductionmentioning

confidence: 99%

“…In addition, S and Z alleles are determined by the allele-specific genotyping method in order to test possible S and Z variants not correctly characterized by phenotyping. If these variants are not detected, exonic sequencing of the SERPINA1 gene is performed 17. This method consists of amplification of the four encoding exons of the SERPINA1 gene by PCR followed by Sanger automated sequencing.…”

Section: Introductionmentioning

confidence: 99%

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Application of a diagnostic algorithm for the rare deficient variant Mmalton of alpha-1-antitrypsin deficiency: a new approach

Belmonte

Barrecheguren

López-Martínez

et al. 2016

COPD

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Background and objectivesAlpha-1-antitrypsin deficiency (AATD) is associated with a high risk for the development of early-onset emphysema and liver disease. A large majority of subjects with severe AATD carry the ZZ genotype, which can be easily detected. Another rare pathologic variant, the Mmalton allele, causes a deficiency similar to that of the Z variant, but it is not easily recognizable and its detection seems to be underestimated. Therefore, we have included a rapid allele-specific genotyping assay for the detection of the Mmalton variant in the diagnostic algorithm of AATD used in our laboratory. The objective of this study was to test the usefulness of this new algorithm for Mmalton detection.Materials and methodsWe performed a retrospective revision of all AATD determinations carried out in our laboratory over 2 years using the new diagnostic algorithm. Samples with a phenotype showing one or two M alleles and AAT levels discordant with that phenotype were analyzed using the Mmalton allele-specific genotyping assay.ResultsWe detected 49 samples with discordant AAT levels; 44 had the MM and five the MS phenotype. In nine of these samples, a single rare Mmalton variant was detected. During the study period, two family screenings were performed and four additional Mmalton variants were identified.ConclusionThe incorporation of the Mmalton allele-specific genotyping assay in the diagnostic algorithm of AATD resulted in a faster and cheaper method to detect this allele and avoided a significant delay in diagnosis when a sequencing assay was required. This methodology can be adapted to other rare variants. Standardized algorithms are required to obtain conclusive data of the real incidence of rare AAT alleles in each region.

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Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Application of a diagnostic algorithm for the rare deficient variant Mmalton of alpha-1-antitrypsin deficiency: a new approach

Belmonte

Barrecheguren

López-Martínez

et al. 2016

COPD

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“…It can only test for those variants for which PCR primers are available. 52,53 There are now methods available to test multiple alleles covering more than 99% of patients with abnormal alleles. 54 These tests can be undertaken on buccal swabs or dried blood spots.…”

Section: Has Screening For Aatd In Patients At Risk Shown To Improve mentioning

confidence: 99%

Diagnosis and treatment of lung disease associated with alpha one‐antitrypsin deficiency: A position statement from the Thoracic Society of Australia and New Zealand*

et al. 2020

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AATD is a common inherited disorder associated with an increased risk of developing pulmonary emphysema and liver disease. Many people with AATD‐associated pulmonary emphysema remain undiagnosed and therefore without access to care and counselling specific to the disease. AAT augmentation therapy is available and consists of i.v. infusions of exogenous AAT protein harvested from pooled blood products. Its clinical efficacy has been the subject of some debate and the use of AAT augmentation therapy was recently permitted by regulators in Australia and New Zealand, although treatment is not presently subsidized by the government in either country. The purpose of this position statement is to review the evidence for diagnosis and treatment of AATD‐related lung disease with reference to the Australian and New Zealand population. The clinical efficacy and adverse events of AAT augmentation therapy were evaluated by a systematic review, and the GRADE process was employed to move from evidence to recommendation. Other sections address the wide range of issues to be considered in the care of the individual with AATD‐related lung disease: when and how to test for AATD, changing diagnostic techniques, monitoring of progression, disease in heterozygous AATD and pharmacological and non‐pharmacological therapy including surgical options for severe disease. Consideration is also given to broader issues in AATD that respiratory healthcare staff may encounter: genetic counselling, patient support groups, monitoring for liver disease and the need to establish national registries for people with AATD in Australia and New Zealand.

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“…При генотипировании выявляются специфические мутации гена ААТ, прежде всего -S и Z. При использовании данного метода применяются принципы полимеразной цепной реакции (ПЦР), при этом также могут выявляться редкие и нулевые варианты, например MMalton [35][36][37]. Однако таким путем можно определить только известные дефекты последовательностей, каждая из которых требует специфических праймеров.…”

Section: лабораторная диагностика и последовательность обследованияunclassified

Diagnosis and treatment of pulmonarydisease in α1-antitrypsin deficiency: a statement of European Respiratory Society

Редакционная¹

2018

Pulʹmonologiâ (Mosk.)

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Alfa-1-antitrypsin deficiency (AATD) is the most common hereditary disorder in adults. It is associated with an increased risk of developing pulmonary emphysema and liver disease. The lung injury in AATD is closely associated with smoking, but progressive lung disease could occur even in never-smokers. A number of individuals with AATD remain undiagnosed and therefore do not receive appropriate care and treatment. The most recent international document on AATD was the joint statement of the American Thoracic Society and the European Respiratory Society published in 2003. Thereafter, there has been a continuous development of novel, more accurate and less expensive genetic diagnostic methods. Furthermore, new outcome parameters have been developed and validated for use in clinical trials and a new series of observational and randomized clinical trials have provided more evidence concerning the efficacy and safety of augmentation therapy, the only specific treatment available for the pulmonary disease associated with AATD. As AATD is a rare disease, it is important to createnational and international registries and to collect information prospectively about the natural history of the disease. Management of AATD patients must be supervised by national or regional expert centres and inequalities in access to therapies across Europe should be addressed.

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Rapid detection of Mmalton α1-antitrypsin deficiency allele by real-time PCR and melting curves in whole blood, serum and dried blood spot samples

Cited by 11 publications

References 19 publications

Application of a diagnostic algorithm for the rare deficient variant Mmalton of alpha-1-antitrypsin deficiency: a new approach

Application of a diagnostic algorithm for the rare deficient variant Mmalton of alpha-1-antitrypsin deficiency: a new approach

Diagnosis and treatment of lung disease associated with alpha one‐antitrypsin deficiency: A position statement from the Thoracic Society of Australia and New Zealand*

Diagnosis and treatment of pulmonarydisease in α1-antitrypsin deficiency: a statement of European Respiratory Society

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