Identification of Three Novel Substitutions in the Erythroid Promoter of the HMBS Gene: A New Erythroid Variant of Human Acute Intermittent Porphyria?

Porphyrias are inherited disorders in the biosynthesis of heme. Acute intermittent porphyria (AIP), the most common form, is autosomal dominant and it is characterized by recurrent attacks of abdominal pain, gastrointestinal dysfunction, and neurologic disturbances. AIP is caused by molecular defects in the hydroxymethylbilane synthase gene (HMBS) causing a partial deficiency of the third enzyme of the heme biosynthetic pathway. This gene maps to chromosome 11q23.2 with a total of 15 exons. Two distinct promoters direct the synthesis of housekeeping and erythroid specific mRNAs by alternative splicing. The housekeeping promoter is located upstream the exon 1 while the erythroid promoter include a portion of intron 1, the exon 2 and the intron 2. The exon 1 and the exons 3 to 15 generate the housekeeping mRNA while the exons 2 to 15 generate the erythroid mRNA giving rise to the housekeeping HMBS isoform and to the erythroid HMBS isoform respectively. In the classic form of AIP, both the housekeeping and the erythroid HMBS isoforms are deficient since the must common molecular defects involve the common region of the gene from exon 3 to exon 15. Approximately 5% of AIP patients have normal levels of the erythroid HMBS isoform and defects in the housekeeping promoter or in the exon 1 which is specific for the housekeeping HMBS isoform, causing the ‘non-erythroid variant’ of AIP. So far no mutations are known in the erythroid promoter. In this study we searched for molecular defects in HMBS gene in three Italian patients with typical clinical and biochemical signs of AIP. The diagnosis was based on elevated urinary excretion of porphyrinic precursors and reduced erythrocyte HMBS activity. The entire HMBS gene has been amplified by PCR and submitted to direct automated sequencing: no mutations known as responsible for classic form of AIP have been identified. However, each of three patients had a substitutions in the erythroid promoter of HMBS gene that could justify the reduced erythrocyte HMBS activity: c.34–115 C>A, c.34–126 G>C and c.34–156G>A. In order to establish if these substitutions were polymorphisms, more than 200 alleles from Italian normal subjects were sequenced and none of them revealed the substitutions. These three mutations, located specifically in the intron 2, could affect the normal splicing of exon 3 causing an abnormal mRNA, to give rise to the classical form of AIP. The mRNA analysis, however, didn’t reveal any abnormal mRNA. Moreover real time experiments in the lymphocytes showed a 100% expression of housekeeping HMBS mRNA excluding the non-sense mediated decay mechanism. These data suggest that these mutations could affect the normal function of the erythroid promoter only, causing an erythroid variant of human acute intermittent porphyria. Further expression studies are in progress.