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Expression and Characterization of Six Mutations in the Protoporphyrinogen oxidase gene among Finnish Variegate Porphyria Patients

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Abstract

Background

Variegate porphyria (VP) is an inherited disorder of heme biosynthesis that results from a partial deficiency of protoporphyrinogen oxidase (PPOX). Patients with VP may experience acute neurovisceral attacks and cutaneous photosensitivity. To date we have characterized 109 VP patients representing 19 VP families in the Finnish population of 5 million, both biochemically and clinically.

Materials and Methods

Mutations were identified by direct sequencing of the patients’ genomic DNA. The effect of the mutations was determined by sequencing the reverse transcriptase polymerase chain reaction (RT-PCR) product amplified from total RNA extracted from the patients’ lymphoblast cell lines and expressing the mutations in E. coli and COS-1 cells.

Results

Of the six mutations identified in the PPOX gene, three mutations (IVS2-2a→c, 338G→C, and 470A→C) caused splicing defects, one produced a frameshift (78insC) and two mutations (R152C and L401F) caused amino acid substitutions. In RT-PCR, the IVS2-2a→c mutation caused a retention of a 36-bp fragment in the 3′ end of intron 2, the 338G→ C mutation caused an exon 4 deletion, and the 470A→ C mutation caused an exon 5 deletion with retention of a 19-bp fragment of the 3′ end of intron 5. In both prokaryotic and eukaryotic expression systems, the PPOX activities of five mutants were decreased to 0–5% of the normal activity.

Conclusions

This study describes five novel mutations and one earlier described major mutation among Finnish VP patients. All mutations produced detectable transcripts, but resulted in decreased PPOX activity confirming the causality of the mutations and the biochemical defects in these patients.

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Acknowledgments

We thank medical students Ms. Jamelah Tucker and Ms. Katri Kuusisto for their excellent assistance in sequencing. This study was supported by grants from the Magnus Ehrnrooth Foundation, the Finnish Cultural Foundation, the Research Funds and the Clinical Research Institute of the Helsinki University Central Hospital, the Biomedicum Helsinki Foundation, and the University of Helsinki.

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Authors and Affiliations

  1. Department of Medicine, Division of Endocrinology, University Central Hospital of Helsinki, Helsinki, Finland

    Mikael von und zu Fraunberg & Raili Kauppinen

  2. Department of Clinical Chemistry, University Central Hospital of Helsinki, Helsinki, Finland

    Raimo Tenhunen

Authors
  1. Mikael von und zu Fraunberg
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  2. Raimo Tenhunen
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  3. Raili Kauppinen
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Correspondence to Mikael von und zu Fraunberg.

Additional information

Mutation R152C partly described earlier in Acta Haematologica (1997) 98(suppl. 1) 1–128 (Abstr.)

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von und zu Fraunberg, M., Tenhunen, R. & Kauppinen, R. Expression and Characterization of Six Mutations in the Protoporphyrinogen oxidase gene among Finnish Variegate Porphyria Patients. Mol Med 7, 320–328 (2001). https://doi.org/10.1007/BF03402215

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