Analysis of genetic variants in the 5’ regulatory region of the ALAS1 gene in South African patients with Variegate Porphyria (VP)

The porphyrias are a group of genetic disorders arising from mutations in either one of the final seven genes encoding the haeme synthesis enzymes. These disease-causing mutations lead to an enzyme deficiency that disrupts normal haeme production, resulting in clinical features due to the subsequent accumulation of porphyrin precursors. Like most of the porphyrias, variegate porphyria (VP) is characterized by high interand intrafamilial clinical variability, with no apparent genotype-phenotype correlation. The delta-aminolevulinate synthase-1 gene (ALAS1) is an apparent candidate gene to explain the variable clinical expression observed in VP, since it encodes the first and rate-determining enzyme of haeme synthesis. Several studies have defined important regulatory elements for the human-, ratand chicken ALAS1 gene that regulate expression patterns of this gene. It was hypothesized that in VP individuals, variants within/near critical regulatory sites might alter the transcription rate of this gene, and consequently increase/decrease the amount of haeme precursors accumulating as a result of the defective haeme synthesis enzyme. The aim of this study was to identify genetic variants that could influence gene expression in the proximal promoter area of the ALAS1 gene, as well as the two ALAS1-drug responsive enhancer sequences (ADRES) located further upstream. DNA (2133 bp per patient) of 19 clinically defined VP patients was analysed by polymerase chain reaction (PCR) and semiautomated DNA sequencing. Subsequently, in silico analyses using appropriate software programs, and in vitro studies using the luciferase reporter system, were performed to investigate the functionality of the identified variants on ALAS1 gene transcription. Two novel single nucleotide polymorphisms (SNPs), a C>T transition at nucleotide -853 and a T>A transversion at nucleotide position -1253, were identified in three VP patients from a single family. No correlation between the sequence variants and clinical symptoms could be observed, suggesting that these two variants are not definitive factors in determining the variable expression of VP. However, due to the small sample size of the VP cohort used, the possibility of variation in ALAS1 gene expression contributing to the complex process of phenotypic presentation in VP and other porphyrias could not be excluded. In silico analysis revealed that the -853C/T variant is located within three bp from a potential half-palindromic estrogen response element (ERE), the estrogen receptor (ERα) binding site, while the -1253T/A variant is located 3’ to a putative non-consensus stimulatory protein-1 (Sp1) site. For in vitro analyses, ALAS1 promoter mutation constructs were generated and cotransfection experiments performed with an ERα-expression vector in HepG2 cells. Results revealed that the ALAS1 wild-type promoter is transcriptionally up-regulated in response to estrogen stimulation and that ERα is required for the observed enhanced activity. Cotransfections with the promoter mutation constructs indicated that the -853T allele amplifies the effect of estrogen, whereas the -1253A allele reduced transcriptional activity irrespective of the presence of estrogen. This study provides evidence for a novel mechanism of haeme regulation via ALAS1 in response to estrogen. These in vivo and in silico findings may also contribute to a better understanding of the variable sympomatic expression of the porphyrias, such as VP, with possible alleviating and aggravating effects mediated by the -1253A and -853T variants respectively.