Porfiria cutanea tardia: Deteccion de una nueva mutacion en la uroporfirinogeno decarboxilasa mediante rt-pcr y secuenciacion directa

Porphyria Cutanea Tarda (PCT) and Hepatoerythropoietic Porphyria (HEP) are characterized by a deficiency in the activity of Uroporphyrinogen decarboxyiase (URO-D), the fifth enzyme in the heme pathway, which catalises the sequential decarboxylation of Uroporphyrinogen to Coproporphyrinogen.There are two types of PCT: sporadic (S-PCT) and familial (F-PCT).The former is an acquired disorder, whereby URO-D activity is only diminished in the liver while the latter is a hereditary dominant disorder where URO-D activity is 50% reduced in all tissues. HEP is a recessive disease where URO-D activity is diminished to 5-10% of normal value. In both porphyrias accumulation of highly carboxilated porphyrins produces the characteristic cutaneous photosensitivity. Human URO-D gene maps in chromosomel (1p34) and comprises 10 exons spread on 3 kb. UROD mRNA has 1,197 bases which codifies a protein of 367 aminoacids.Ten mutations in URO-D gene have been already identified causing F-PCT or HEP. In this work we describe the case of an argentinean patient with the classical symptoms of PCT: high urinary porphyrins levels (8,909; NV=20-250 μg/24 hrs), with the characteristic excretion pattern and an elevated plasma porphyrin index (7.48; NV < 1.30). Erythrocytic URO-D activity was measured employing Uroporphyrinogen as substrate and the porphyrins obtained were analized by HPLC. The URO-D value of the patient was 7.33 U/ml RBC (45% of normal value= 16.35 ±3.56 U/ml RBC). Lymphocytes were separated first from fesh whole blood by density gradient, total RNA was isolated using the commercial reagent RNA _zol ™B (BIOTECX). Then RT-PCR reactions were carried out employing M-MLV RT and oligo(dT) for RT andTaq DNA polymerase and the adequate primers for amplification. The product was purified employing Wizard™ (PROMEGA) minicolums and it was sequenced by Sanger methodology using a modified Taq polymerase (fmol PROMEGA) or sequence after asymétrie PCR to obtain both single strands separately. Two products were obtained in the RT-PCR; both bands were cut from 2% agarose gel and eluted in TE buffer. They were then sequenced, showing that in the small band the 67 bases corresponding to exon 9 were missing. DNA genomic analysis showed the transition G→A in the last base of exon 9 (E314E) causing its skipping. Furthermore, due to the joint of exons 8 and 10 the reading frame was changed, leading to the formation of a stop codon and the synthesis of a truncate protein with the losing of about 20% of its aminoacids.