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Annals of Clinical & Laboratory Science 32:107-113 (2002)
© 2002 Association of Clinical Scientists

Single-Strand Conformational Polymorphism and Denaturing Gradient Gel Electrophoresis in Screening for Variegate Porphyria: Identification of Two New Mutations

James G. Donnelly, Sarah Detombe and J. Thomas Hindmarsh
Division of Biochemistry, The Ottawa Hospital, and Department of Pathology and Laboratory Medicine, University of Ottawa, Ottawa, Ontario, Canada

Address correspondence to J. Thomas Hindmarsh, M.D., Division of Biochemistry, The Ottawa Hospital, 501 Smyth Road, Ottawa, Ontario, Canada KiH 8L6; tel 613 737 8312; fax 613 737 8315; e-mail jhindmarsh{at}ottawahospital.on.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Single-strand conformational polymorphism and denaturing gel electrophoresis were used to screen for mutations in the protoporphyrinogen oxidase gene (PPOX) of three patients with clinically and biochemically proven variegate porphyria in order to select genomic regions for specific DNA sequence analysis. Two previously undescribed mutations were identified: PPOX1423–1426-delATCT and PPOX2272insG. Denaturing gel electrophoresis was able to discern the point mutation in exon 5 (PPOX2272insG) of the PPOX gene. Once an index individual has been identified, single-strand conformational polymorphism and denaturing gel electrophoresis techniques are useful to identify family members who may be unaffected carriers. Such identification can help potential cases to avoid medications and other triggers that could precipitate acute porphyric attacks.

(received 14 July 2001; accepted 9 November 2001)

Keywords: variegate porphyria, urine porphyrins, stool porphyrins, mutation, protoporphyrinogen oxidase


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Variegate porphyria is one of the four classes of acute hepatic porphyrias [1]. It is inherited as an autosomal dominant trait and its clinical manifestations include abdominal pain, neurological damage, and photo-sensitive dermatitis. Variegate porphyria is related to partial deficiency in several tissues of a mitochondrial enzyme, protoporphyrinogen oxidase [E.C.1.3.3.4], which is encoded by the PPOX gene on chromosome 1q22–23 [2–4].

Clinical features cannot differentiate the acute porphyrias. The dermatologic features are identical in variegate porphyria and hereditary coproporphyria and the neurovisceral features are similar in all of the acute porphyrias. Biochemical diagnosis of variegate porphyria is usually conclusive in a well-established case [5], but porphyrin metabolite excretion can occasionally be normal in long-dormant cases. Moreover, porphyrin analysis is often normal in the large proportion of persons who inherit the enzyme defect but have never been symptomatic.

Enzyme analysis would identify all the above but, since protoporphyrinogen oxidase is a mitochondrial enzyme, it is not present in erythrocytes; liver tissue or fibroblast culture is therefore necessary [6]. Genetic analysis is useful in family studies and for identifying potential cases, such as the children of a patient, provided the patient has an identifiable genetic abnormality.

A variety of techniques can be used to screen for genetic mutations in this disease. Single-strand conformational polymorphism (SSCP) is widely used to detect single nucleotide changes in small DNA fragments. In this technique, samples of genomic DNA from patients and controls are amplified using primers for the appropriate exons and their flanking regions by reverse transcription. The PCR product is denatured to separate the strands, which are then run on a non-denaturing gel. Strands that differ by as little as a single base-pair have mobilities in the gel that are clearly different from the normal control and appropriate samples can thereby be selected for molecular sequencing. The sensitivity of this technique is inversely proportional to fragment size. It favors the scanning of shorter segments of DNA and works best with PCR products that are approximately 250 base pairs in length.

Denaturating gradient gel electrophoresis (DGGE) is based upon the electrophoretic mobility of a double-stranded DNA molecule through linearly increasing concentrations of a denaturing agent (formamide). By denaturing and then renaturing the DNA strands, heterogeneous mismatched duplex strands are formed. A DNA duplex with a mismatched pair has a different mobility than a fully matched double-stranded fragment because of the effect of the mismatch on the secondary structure of the molecule. We used these techniques to select samples for molecular sequencing from three patients with clinically apparent and biochemically proven variegate porphyria.

Our patients were a sister and brother (Patients 1 and 2) and an unrelated subject (Patient 3). Patient 1, a 40-yr-old female Caucasian, had mild recurrent abdominal pain since puberty, as well as light sensitive skin reactions related to taking estrogens. Patient 2, her 44-yr-old brother, had suffered for many years from bullous skin lesions on the hands and to lesser extent, face, which were worse in the summer. Patient 3 was a 49-yr-old female Caucasian with mild recurrent skin lesions typical of photosensitive porphyria since age 18 yr. The patients all had a family history of likely cases of porphyria .


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Acknowledgements
 References
 
DNA Isolation and PCR Amplification.  Genomic DNA from leukocytes of patients and controls was isolated for PCR using the WizardTM Prep System (Promega, Inc., Madison, WI). PCR was performed using reagents and Taq polymerase from Applied Biosystems (Mississauga, Ontario, Canada). Primers were obtained from BioCorp (Montreal, Quebec, Canada). The genomic DNA was amplified for exons and intron boundaries of all PPOX exons (Table 1Go). This includes the entire coding sequence of the gene and the canonical regions required for proper splicing. An aliquot of each PCR product was analyzed by electrophoresis on 2% agarose in 0.5X tris-borate EDTA buffer, pH 8.3 (TBE) to verify the DNA product size.


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  		Table 1. PCR primers that were used to investigate the protoporphyrinogen oxidase (PPOX) gene [13,14].
 
Mutation Screening.  SSCP was performed using a modification of the method of Schreiber et al [7]. Aliquots of the PCR products were heated to 100°C for 7 min in 37.5% formamide and 7.5% glycerol to permit denaturation. The DNA was separated by electrophoresis on 7% acrylamide gel in 0.5X TBE buffer at 300–350V for 2–3 hr.

Heteroduplex strands were formed using a modification of the method of Tchernitchko et al [8]. Heteroduplex strands were formed by heating the PCR products to 68°C for 1 hr using 20% ethylene glycol and 30% formamide. DNA was separated using denaturing gels with a TBE buffer gradient at ambient temperature [8]. Silver staining was used to visualize both SSCP and DGGE [9].

Direct Sequence  Analysis. Mutations observed using SSCP were sequenced using the specific primers for the exon that contained the mutation. Sequencing was performed with the ThermoSequenaseTM radiolabelled terminator sequencing kit (United States Biochemical Corp., Cleveland, OH).

Biochemical Analyses.  Fecal and urine porphyrins were measured by reversed-phase high performance chromatography [5]. In Patients 2 and 3, an earlier version of our method was used that did not quantify uroporphyrin or the hepta-, hexa-, and penta-carboxyl intermediates in feces, nor did it separate the I and III isomers of uroporphyrin or coproporphyrin in urine or the I and III isomers of coproporphyrin in feces. Urine delta-aminolevulinic acid (ALA) and porphobilinogen (PBG) were measured by ion exchange chromatography (Bio-Rad Co., Mississauga, Ontario, Canada). Erythrocyte porphobilinogen deaminase (hydroxymethylbilane synthase) was measured by our modification of the method of Piepkorn et al [10], using ALA as substrate. Plasma fluorescence scanning was performed by the method of Poh-Fitzpatrick [11], with the plasma sample diluted in phosphate buffer (pH 7.4) and scanned by a spectrofluorimeter (excitation 405 nm, emission 550–700 nm); in some cases of variegate porphyria, a protein-porphyrin complex produces an emission peak at 625 nm.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Acknowledgements
 References
 
Two separate mutations were identified in the unrelated patients. The sister and brother (Patients 1 and 2) had a mutation in exon 3, PPOX 1423–1426delATCT, which is predicted to result in a prematurely truncated form of PPOX. This mutation was observed using SSCP (Fig. 1AGo). The mutation found in Patient 3 was PPOX2272insG. This frameshift mutation was also predicted to cause premature truncation of the PPOX protein peptide. Interestingly, this mutation was not detected by SSCP (Fig. 1BGo).



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  		Fig. 1. Electrophoretic patterns that document mutations of the protoporyphyrinogen oxidase (PPOX) gene in two patients with variegate poryhyria.

Panel A (PPOX exon 3, Patient 1). The single-strand conformational polymorphism (SSCP) pattern of a silver-stained acrylamide gel is shown in the left section. Lane M: marker DNA; lane C: pooled DNA from unaffected individuals; lane P: DNA from Patient 1. The radiographic pattern of a direct sequencing gel is shown in the right section; G, A, T, and C indicate the terminal nucleotide in the respective lanes. The mutation PPOX 1423–1426delATCT is marked by an arrow. Patient 2, sibling of Patient 1, had identical results and the same mutation.

Panel B (PPOX exon 5, Patient 3). The SSCP pattern in the left section and the denaturing gradient gel electrophoretic (DGGE) pattern in the middle section are silver-stained acrylamide gels. Lane M: marker DNA; lanes C: pooled DNA from unaffected individuals; lanes P: DNA from Patient 3. The radiographic pattern of a direct sequencing gel is shown in the right section; G, A, T, and C indicate the terminal nucleotide in the respective lanes. The mutation PPOX2272insG is marked by an arrow.

 
Table 2Go lists the fecal and urine porphyrin excretions of Patients 1, 2, and 3; they all had a pattern typical of variegate porphyria: elevation of stool protoporphyrin and (to a lesser extent) coproporphyrin, together with an elevation of urine coproporphyrin (with a predominance of the III isomer of coproporphyrin in urine and stool in the only subject in which they were fractionated, Patient 1). The plasma fluorescence peak, typical of variegate porphyria, was present in Patient 2, absent in Patient 1, and not measured in Patient 3. Patients 1 and 2 had experienced elevations of ALA and PBG in urine at some time during their clinical courses. All three cases had normal levels of erythrocyte porphobilinogen deaminase (hydroxymethylbilane synthase) in keeping with their diagnosis.


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  		Table 2. Measurements of porphyrin metabolism in three patients with variegate porphyria.
 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Acknowledgements
 References
 
The protoporphyrinogen oxidase gene, whose mutations are responsible for variegate porphyria, contains 13 exons and ~5 kilobases. The disease is more common in South Africa where most patients have the R59W mutation [12]. Elsewhere, the disease is heterogeneous and, to date, more than 79 mutations have been observed in PPOX [12–30]. The largest study emanated from Nordmann and Deybach’s laboratory [14]. These workers found that mutations in French patients seem to differ from those in British patients, despite the overlapping ancestry of the two countries. Whatley et al [14] speculated that this might imply a relatively recent origin of the mutations. We report two additional mutations in three patients, some of whose forebears came from Britain.

Clinical features in conjunction with biochemical assessment are almost always sufficient to make a diagnosis of symptomatic variegate porphyria. However, in dormant cases the biochemical abnormalities may be minimal or absent (except for the assay of protoporphyrinogen oxidase, which is impractical for most investigators). In these circumstances, molecular diagnosis may be helpful, particularly if the patient has one of the previously described mutations. Molecular techniques are also useful in studies of asymptomatic relatives to identify those at risk for developing the disease, although it must be noted that only ~10% of these will ever develop symptoms.

In this study, DGGE proved superior to SSCP in identifying patients with mutations in the PPOX gene. The increased sensitivity of DGGE is likely due to the larger size (duplex) product used in the separation. A single nucleotide insertion or point mutation may not disrupt the folding of a PCR product enough to cause it to migrate differently using SSCP. Premature truncation within the peptide strand at either exon-3 or exon-5 apparently suppresses the catalytic activity of the PPOX enzyme, as reflected in the biochemical and clinical features of these patients. In either mutation, less than 50% of the peptide strand is transcribed.

Both multiple and single insertion mutations typically allow the transcription of one or more incorrect amino acids when a frameshift occurs. A stop codon usually prevents substantial expression of an inappropriate protein: one that can alter normal cellular function. Even if the catalytic domain is transcribed, premature truncation can result in improper folding, a shortened half-life, and an absence of catalytic activity. Often the peptide arising from the mutated gene will have a shortened half-life or not be delivered to its proper cellular location.

Except for the South African PPOX mutation, the other mutations all appear to be private. No mutational hotspots have been identified in the PPOX gene. One should recognize that the heme pathway is sensitive to mutations. Heme is an ancient molecule that is essential for life. Humans are dependent upon two functional copies of the genes required for heme synthesis. There appears to be no selective advantage in having a mutation that reduces heme synthesis; mutations that decrease enzymatic activity in this pathway are all likely to be harmful.

The pathogenesis of the disease usually permits transmission of the mutation to offspring. The severity of the disease or its penetrance probably have less to do with the nature of the mutation and more to do with environmental pressures that are superimposed on the mutational change. Together, these factors may affect the tolerance of the patient to decreased activity in the heme synthesis pathway.

In conclusion, the capability of detecting family members who are at risk of porphyria is benefited by simple PCR and electrophoretic techniques. This approach is in contrast to the traditional biochemical assessments, which can have unpredictable outcomes depending upon the metabolic status of the patient.


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
References
 
This study was partly funded by the Research and Development Fund of the Department of Pathology and Laboratory Medicine of The Ottawa Hospital, Ottawa, Ontario, Canada.

Some of this material was previously reported in abstract form [31,32].


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

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