Three novel mutations in the coproporphyrinogen oxidase gene

✍ Scribed by Jérôme Lamoril; Jean-Charles Deybach; Hervé Puy; Bernard Grandchamp; Yves Nordmann

• Publisher: John Wiley and Sons
• Year: 1997
• Tongue: English
• Weight: 109 KB
• Volume: 9
• Category: Article
• ISSN: 1059-7794
• DOI: 10.1002/(sici)1098-1004(1997)9:1<78::aid-humu17>3.0.co;2-m

✦ Synopsis

Three unrelated patients with HC were studied. The diagnosis of coproporphyria was based on the observation of typical clinical manifestations, increased excretions of Ç-aminolevulinic acid and porphobilinogen in urine and coproporphyrin III in both feces and urine, and 50% decreased COX activity in lymphocytes (Table 1). The patients were screened for the presence of mutations in the coding sequence and exon-intron boundaries of COX gene (exons 1-7) by direct sequencing of polymerase chain reaction (PCR)-amplified fragments of the gene. Primers for PCR amplification and sequencing were selected from the published COX sequence (Table 2). (Delfau-Larue et al., 1994). Genomic DNA was extracted from peripheral blood as previously described (Higuchi, 1989). Genomic DNA sequences were amplified with specific primers (Table 2), 20 pmol of each in 50 ãl of PCR solution containing 1 unit of Taq polymerase (Beckmann, Fullerton, CA), 50 mmol/L KCl, 10 mmol/L Tris-HCl pH 8.5, 1.5 mmol/ L MgCl 2 , 200 mmol/L of each dNTP. The reactions were performed in a DNA thermocycler (Hybaid, Teddington, UK) as follows: 35 cycles of denaturation at 94°C for 30 secs, annealing at specific temperature (Table 2) for 30 secs and elongation at 72°C for 1 min, followed by a final extension at 12°C for 10 min.

The PCR products were purified with Wizard PCR preps DNA purification system (Promega-Biotech, Madison, WI) then direct PCR sequencing was performed using the fmol DNA sequencing kit