Severe alpha-1 antitrypsin deficiency in composite heterozygotes inheriting a new splicing mutation QOMadrid.

Beatriz Lara,Cristina Hernández-Moro,Maria Teresa Martínez,Ilaria Ferrarotti,Irene Vazquez,Francisco Rodriguez-Frias,Manuel Posada,Laura Perez,Javier Alonso,Ignacio Blanco,Eladio A Velasco,Beatriz Martínez-Delgado

doi:10.1186/s12931-014-0125-y

Abstract

BackgroundSevere Alpha-1 Antitrypsin (AAT) deficiency is a hereditary condition caused by mutations in the SERPINA1 gene, which predisposes to lung emphysema and liver disease. It is usually related to PI*Z alleles, and less frequent to rare and null (QO) alleles. Null-AAT alleles represent the end of a continuum of variants associated with profound AAT deficiency and extremely increased risk of emphysema.MethodsA family with severe AAT deficiency was analyzed to achieve genetic diagnosis. The complete exons and introns of the SERPINA1 gene were sequenced and transcriptional analysis by RT-PCR was performed to characterize the effect of splicing variants found in the patients. In addition, a minigene MGserpa1_ex1b-1c was cloned into the pSAD vector to in vitro investigate the independent impact of variants on splicing process.ResultsWe report a new identified null allele (PI*QOMadrid) in two adult siblings with practically no detectable serum AAT. The PI*QOMadrid allele consist of a duplication of the thymine (T) in position +2 of the donor splice site of exon 1C (+2dupT). In these two subjects, PI*QOMadrid occurred in compound heterozygote combination with the previously described variant PI*QOPorto. Both QOMadrid and QOPorto variants are located very close together in a regulatory region of the SERPINA1 gene. Analysis of transcripts revealed that QOMadrid variant prevented the expression of transcripts from exon 1C, and then normally spliced RNA products are not expected in the liver of these patients. In addition, aberrant splicing patterns of both variants were clearly distinguished and quantified by functional in vitro assays lending further support to their pathogenicity.ConclusionFinding pathogenic mutations in non-coding regions of the SERPINA1 highlight the importance that regulatory regions might have in the disease. Regulatory regions should be seriously considered in discordant cases with severe AAT deficiency where no coding mutations were found.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-014-0125-y) contains supplementary material, which is available to authorized users.

Highlights

Severe Alpha-1 Antitrypsin (AAT) deficiency is a hereditary condition caused by mutations in the SERPINA1 gene, which predisposes to lung emphysema and liver disease
Other previously described AAT null alleles have been reported in Spain [10], we describe the first new AAT null allele in Spain, in a Caucasian family from Madrid, designated as QOMadrid, as this city was the place of birth and residence of the index-case, as well as his three siblings and parents
Computational predictions By using different prediction methods integrated in Alamut 1.3 software we evaluated splice signal detection (SpliceSiteFinder-like, MaxEntScan, GeneSplicer, Human Splicing Finder or Known constitutive signals) and Exonic Splicing Enhancers exonic splicing enhancers (ESE) binding site detection (ESEFinder) comparing reference and mutated sequences

Summary

Introduction

Severe Alpha-1 Antitrypsin (AAT) deficiency is a hereditary condition caused by mutations in the SERPINA1 gene, which predisposes to lung emphysema and liver disease. Null-AAT alleles represent the end of a continuum of variants associated with profound AAT deficiency and extremely increased risk of emphysema. Severe AAT deficiency is an inherited condition characterized by AAT serum levels below 35% (or 50 mg/dL) the normal value. The protein is codified at the protease inhibitor locus (14q32.1), by the SERPINA1 gene, which is organized into three non-coding exons Over 80% of AAT is synthesized in the liver, other cell types such as blood monocytes, neutrophils, macrophages, pancreas, endothelium, enterocytes, lung alveolar and some cancer cells are capable of secreting additional quantities [6,7]. Transcriptional regulation occurs in at least two different sites within the gene: the hepatocyte promoter located upstream the transcription start site of exon 1C, and the monocyte promoter located upstream of exon 1A [8]

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