Actualización del algoritmo de diagnóstico de laboratorio del déficit de alfa-1-antitripsina: incorporación de la detección genotípica de la variante deficitaria Mmalton y utilización de muestras alternativas a la sangre total

Abstract

Alpha-1-antitrypsin deficiency (AATD) is a genetic disorder characterized by low serum levels of alpha-1-antitrypsin protein (AAT) and a high risk of developing early onset emphysema and liver disease. About 125 allelic variants of the AAT gene have been described. The most common normal AAT allele is the M variant, and the most frequent deficient variants are Z and S. However, another rare deficient variant, called Mmalton, which causes a deficiency similar to variant Z, is considered to be the second cause of severe AATD in Spain. This variant is difficult to detect with the common diagnostic techniques (serum AAT measurement and phenotype characterization). This fact has contributed to a misclassification of this variant and the subsequent underestimation of its real prevalence in the population. Thus, we designed an allele-specific genotyping technique for the detection of the Mmalton variant and we tested its applicability using whole blood, DBS (dried blood spot) and serum samples. Results showed the utility of this technique for the rapid and cost-effective characterization of a deficiency variant with a difficult detection. Moreover, this method could be adapted for the study of the most prevalent rare variants in each region. When genotyping is required, DNA from whole blood or DBS sample is necessary. Occasionally these kinds of samples are not available in the laboratory and additional new extraction is required, causing a significant delay in AATD diagnosis. To avoid this, we developed allele-specific genotyping and exonic sequencing of AAT gene protocols using DNA present in serum samples (sample used at the first steps of the diagnosis). These techniques were incorporated to the laboratory diagnostic algorithm, allowing the complete diagnosis using an only kind of sample and thus, avoiding the delay generated when genotyping is necessary. With the purpose of promoting the expansion of screening programs for the identification of AATD individuals, buccal swab samples were assessed as an alternative to DBS samples. This kind of sample is easy to collect, store and deliver and it recovers a large amount of DNA, resulting in an easy genotyping. Results showed these methodologies may be useful to expand AATD screening programs and complete the diagnostic without delay. The algorithm proposed in this study allows a complete AATD diagnosis avoiding two main problems: significant delay when the doctor performs the diagnostic request and the underdiagnosis of the rare AAT variants.

Date of Award	29 Sept 2017
Original language	English
Supervisor	Francisco Rodriguez Frias (Director)