The Modulatory Effects of the Polymorphisms in GLA 5′-Untranslated Region Upon Gene Expression Are Cell-Type Specific

Abstract

Lysosomal α-galactosidase A (αGal) is the enzyme deficient in Fabry disease (FD). The 5′-untranslated region (5′UTR) of the αGal gene (GLA) shows a remarkable degree of variation with three common single nucleotide polymorphisms at nucleotide positions c.-30G>A, c.-12G>A and c.-10C>T. We have recently identified in young Portuguese stroke patients a fourth polymorphism, at c.-44C>T, co-segregating in cis with the c.-12A allele. In vivo, the c.-30A allele is associated with higher enzyme activity in plasma, whereas c.-10T is associated with moderately decreased enzyme activity in leucocytes. Limited data suggest that c.-44T might be associated with increased plasma αGal activity. We have used a luciferase reporter system to experimentally assess the relative modulatory effects on gene expression of the different GLA 5′UTR polymorphisms, as compared to the wild-type sequence, in four different human cell lines. Group-wise, the relative luciferase expression patterns of the various GLA variant isoforms differed significantly in all four cell lines, as evaluated by non-parametric statistics, and were cell-type specific. Some of the post hoc pairwise statistical comparisons were also significant, but the observed effects of the GLA 5′UTR polymorphisms upon the luciferase transcriptional activity in vitro did not consistently replicate the in vivo observations.

These data suggest that the GLA 5′UTR polymorphisms are possible modulators of the αGal expression. Further studies are needed to elucidate the biological and clinical implications of these observations, particularly to clarify the effect of these polymorphisms in individuals carrying GLA variants associated with high residual enzyme activity, with no or mild FD clinical phenotypes.

Competing interests: None declared

Appendices

Synopsis

The polymorphisms in the α-galactosidase gene (GLA) 5′-untranslated region have cell-type-specific modulatory effects upon gene expression.

Compliance with Ethics Guidelines

This study was conducted according to the applicable national and institutional regulatory and ethical standards. Anonymised male genomic DNA samples were used as the source of the various polymorphic GLA 5′-untranslated region nucleotide sequences assayed in the luciferase reporter system experiments reported herein. The original samples had been obtained and genotyped in prior research projects carried out in the laboratory of molecular genetics of the Department of Genetics, Faculty of Medicine, University of Porto, Portugal, which were approved by the institutional Health Ethics Board and subject to written informed consent. The preservation of the anonymised genomic DNA samples was authorised by the Portuguese Data Protection Authority.

The study protocol did not involve any studies performed with human or animal subjects.

This work is part of Susana Ferreira’s Ph.D. thesis, supervised by João Paulo Oliveira and co-supervised by Carlos Reguenga.

All authors were involved in the conception and design of the study, had full access to all the data and were involved in their analysis and interpretation. Susana Ferreira performed the laboratory work and, as corresponding author, had the final responsibility for the decision to submit the manuscript for publication.

The authors state that none of the material contained in the submitted manuscript has been published previously. Preliminary results of this research project were presented as posters at the European Conference of Human Genetics, Paris, June 2013, and at the Fabry Expert Lounge, Rome, March 2014.

Conflict of Interest Declaration

Susana Ferreira has received unrestricted research grants and funding for research projects from Genzyme Corporation; conference registration fees and travel grants from Genzyme Corporation and Shire Human Genetic Therapies.

Carlos Reguenga declares no conflicts of interest related to the subject matter of this manuscript.

João Paulo Oliveira is member of the European Advisory Board of the Fabry Registry, a global observational registry of patients with Fabry disease sponsored by Genzyme Corporation. He has received unrestricted research grants and funding for research projects from Genzyme Corporation; consulting honoraria and speaker’s fees from Genzyme Corporation; conference registration fees and travel grants from Genzyme Corporation, Shire Human Genetic Therapies and Amicus Therapeutics.