Definition and frequency
In the dual porphyrias, laboratory tests indicate the simultaneous deficiency of two enzymes along the haeme biosynthetic pathway either in one individual or within one family. To date, approximately 15 patients and families with this constellation have been reported (18–33), indicating that these porphyria variants are very rare, although some authors already raised the question 20 years ago whether this peculiar form of porphyria is underdiagnosed (34).
The majority of these dual porphyrias comprise the combined deficiency of uroporphyrinogen decarboxylase (UROD) with either porphobilinogen deaminase (PBGD), coproporphyrinogen oxidase (CPOX) or protoporphyrinogen oxidase (PPOX), respectively (18–26,29,31,32), and this is not surprising because PCT is the most frequent type of porphyria in the world. In three families, the simultaneous deficiency of CPOX and either PBGD (27), uroporphyrinogen III synthase (UROS) (28) or ALAD (33) was detected. In one patient with CEP, the UROS defect was accompanied by a deficiency of UROD (30).
The different types of dual porphyrias reported to date are summarized in Table 3.
Table 3. Chronological order of the different variants of dual porphyrias reported to date
|Authors||Enzyme deficiencies reported||Genetic confirmation||Reference|
|Watson et al.||Uroporphyrinogen decarboxylase and protoporphyrinogen oxidase||No||(18,19)|
|Levine et al.||Uroporphyrinogen decarboxylase and protoporphyrinogen oxidase||No||(20)|
|Day et al.||Uroporphyrinogen decarboxylase and protoporphyrinogen oxidase||No||(21)|
|McColl et al.||Porphobilinogen deaminase and protoporphyrinogen oxidase||No||(22)|
|Doss||Porphobilinogen deaminase and uroporphyrinogen decarboxylase||No||(23)|
|Doss||Porphobilinogen deaminase and uroporphyrinogen decarboxylase||No||(24)|
|Doss||Porphobilinogen deaminase and uroporphyrinogen decarboxylase||No||(25)|
|Sturrock et al.||Uroporphyrinogen decarboxylase and protoporphyrinogen oxidase||No||(26)|
|Nordmann et al.||Coproporphyrinogen oxidase and uroporphyrinogen III synthase||No||(27)|
|Gregor et al.||Coproporphyrinogen oxidase and porphobilinogen deaminase||No||(28)|
|Sieg et al.||Uroporphyrinogen decarboxylase and protoporphyrinogen oxidase||No||(29)|
|Freesemann et al.||Uroporphyrinogen III synthase and uroporphyrinogen decarboxylase||No||(30)|
|Doss et al.||Coproporphyrinogen oxidase and uroporphyrinogen decarboxylase||No||(31)|
|Harraway et al.||Uroporphyrinogen decarboxylase and porphobilinogen deaminase||yes||(32)|
|Akagi et al.||Coproporphyrinogen oxidase and δ-aminolevulinic acid dehydratase||Yes||(33)|
Two types of porphyria within one family
In 1975, Watson et al. for the first time reported on two siblings exhibiting different forms of enzymatic deficiencies affecting haeme biosynthesis. In this family, a 54-year-old woman (designated individual P430) revealed acute neurological attacks and a stool porphyrin excretion pattern indicating VP, whereas her 59-year-old brother (designated individual P431) had never experienced acute attacks and revealed isocoproporphyrin (ISO-COPRO) in the faeces, indicative of PCT. Additionally, several family members showed a biochemical urine and stool porphyrin profile compatible with latent VP. Of note, however, in none of the other family members, ISO-COPRO could be detected in the faeces, raising the question whether, in this family, VP was segregating as an autosomal-dominant trait, whereas individual P431, who exhibited ISO-COPRO in the faeces, was suffering from acquired PCT (18). This problem was then resolved in a later publication from this group in which they reported on the finding of ISO-COPRO in the faeces of a niece of individuals P430 and P431. Based on these data, the authors concluded that both VP and PCT were segregating as independent traits within this family and that the type of PCT present was rather the inherited variant than the acquired form (19).
While in this family the characteristic presence or absence of ISO-COPRO in the faeces of several relatives studied by Watson et al. eventually allowed for an answer to the question which variants of porphyria were present, the authors would have certainly appreciated the possibility of molecular genetic analysis to unequivocally confirm their diagnoses. Unfortunately though, at that time, the genes coding for the different types of porphyrias were not known and, likewise, PCR amplification of specific DNA sections was not available yet.
In the years following the publication from Watson and colleagues, Levine et al. and Day et al. also reported on the coexistence of two distinct types of porphyrias within one family (20,21).
The probably most well-known report on a family with dual porphyria is the one about the so-called Chester porphyria.
In 1985, a new variant of porphyria with autosomal-dominant inheritance was reported from a large kindred residing in Chester, UK, designated Chester porphyria (CP). Affected family members revealed acute porphyric attacks and the biochemical characteristics of both VP and AIP, with some patients revealing overlapping values of porphyrins and porphyrin precursors in the urine and faeces. Additional enzymatic studies showed reduced activity of PBGD and PPOX in individuals with overt disease (22). In 1992, the results of a genome-wide linkage analysis performed in the CP family indicated that a novel gene residing on chromosome 11q23.1 might be involved in the pathogenesis of this novel subtype of dual porphyria (35). Interestingly, this locus did not contain any of the thus far known genes coding for enzymes catalysing major steps in haeme biosynthesis.
In an effort to elucidate the molecular basis of this presumably novel type of porphyria, we obtained DNA samples of 10 individuals from the original CP family, five of whom had been classified as affected and the other five as unaffected, in accordance with previous extensive biochemical and enzymatic studies (22,36). Subsequently, several candidate genes within the candidate interval on 11q23.1 were cloned and screened for mutations. However, these initial efforts were not successful.
Although the original linkage report from Norton et al. excluded the PBGD and PPOX gene as candidates (35), we nevertheless decided to sequence these two genes because the biochemical and enzymatic data published by McColl et al. indicated deficiencies of the encoded enzymes (22). Sequencing analysis of the coding regions of the PPOX gene and its promoter region revealed no mutations. In exon 9 of the PBGD gene, however, we detected a nonsense mutation, designated R149X, that was carried by all affected individuals studied (Fig. 2) (37).
Figure 2. Results of mutation analysis in the Chester porphyria family. The sequence deviation consists of a heterozygous C-to-T transition, indicated by an arrow in the lower panel. This nucleotide change results in a nonsense mutation in exon 9 of the PBGD gene, designated R149X.
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Interestingly, our study revealed that only four of the five individuals from the CP family classified as suffering from overt disease and sent to us for molecular analysis were indeed carriers of the disease-causing mutation R149X. This suggested that the original classification into affected and non-affected individuals in the publications from McColl et al., Norton et al. and Qadiri et al. might have already been somewhat imprecise (22,35,36). Furthermore, this could also explain why the linkage data from Norton et al. erroneously indicated a novel chromosomal locus for CP and why the PBGD gene locus on 11q23.3 was initially excluded on the basis of several recombination events observed in certain members of the CP family (35), assuming that some of the family members revealing the crucial recombination events were perhaps wrongly classified prior to linkage.
Our data show that the individuals from the CP family do not suffer from a novel type of porphyria, but rather from a variant of AIP. It still remains elusive why some individuals revealed the characteristic porphyrin excretion patterns of VP and reduced enzymatic activities of PPOX, likewise indicative of VP.
Interestingly, however, different groups have previously pointed out that biochemical analyses and even the measurement of enzymatic activities in different cells are somewhat imprecise, as a certain overlap between the values measured in patients with overt porphyria, clinically unaffected mutation carriers (the so-called ‘silent’ carriers) and normal control individuals can be found. Thus, the results of biochemical and enzymatic studies in the porphyrias are not always conclusive, sometimes making an accurate diagnosis of the respective type of porphyria difficult if not impossible (16,17).
In support of this notion, several authors found that a coexistent decrease in PBGD activity can be frequently detected in patients suffering from VP who usually exhibit a catalytic deficiency of PPOX solely (38–40). Still, in none of these individuals, the existence of a dual porphyria could be confirmed on the genetic level. Even in a VP family which revealed a concomitant decrease in PBGD activity being as high as 50% of the normal range, no underlying mutation in the PBGD gene was detected and, furthermore, this catalytic deficiency apparently had no clinical consequences (40). Taking these reports into consideration, it might be possible that the accompanying decrease in PPOX activity observed in some members of the CP family is most likely attributable to a phenomenon secondary to the disease-causing genetic defect in the PBGD gene and, thus, has no immediate consequences on the clinical course of the disease.
The results of our studies in the CP family clearly indicate that CP is neither a dual porphyria nor a separate type of porphyria, but rather a variant of AIP. Furthermore, our data also largely exclude the possibility that a hitherto unknown gene is involved in the pathogenesis of the porphyrias.
Confirmation of dual porphyrias by molecular genetic analysis
Until 2006, the diagnosis of dual porphyria in all patients and families reported up to this time was always established on the basis of biochemical measurement of porphyrins and/or porphyrin precursors in urine and faeces and enzymatic assays (18–31). However, in none of these individuals, the diagnosis was confirmed on the molecular genetic level by demonstrating the simultaneous occurrence of two disease-causing mutations in different genes. This might be due to three reasons mainly. First, the fact that PCR-based DNA analysis is a relatively new technique (41) and, thus, the first mutation reports on the porphyrias date from the late 1980s only (42). Second, cDNA and genomic sequences of genes encoding human enzymes involved in haeme biosynthesis were not available until the late 1980s and early 1990s, the first one published being the cDNA sequence of UROD (43). Third, the frequency of type I PCT is approximately three to four times higher than that of type II PCT (1,2) and, therefore, a deficiency of UROD, when encountered in a patient or a family with dual porphyria, might rather be acquired than inherited.
Taking this into consideration, it is understandable that, until recently, there has been no report about a patient with mutations in two genes encoding enzymes of haeme biosynthesis.
In January 2006, two groups independently reported on the first porphyria patients in whom DNA analysis unequivocally confirmed the presence of mutations in two different genes encoding enzymes that catalyse major steps in haeme biosynthesis (32,33).
Harraway et al. reported on a young female patient who developed skin symptoms on the sun-exposed areas of the body and revealed an increased urinary porphobilinogen (PBG) excretion. The initial diagnosis of VP, however, could not be confirmed and biochemical analysis of her urine, faeces and plasma rather indicated a combined deficiency of PBGD and UROD. Subsequent automated sequencing of the PBGD and UROD gene revealed mutations in both genes, confirming the diagnosis of dual porphyria on the molecular genetic level (32).
Akagi et al. studied a male individual who revealed acute porphyric attacks accompanied by an increased urinary excretion of δ-aminolevulinic acid (ALA), PBG and coproporphyrin. Although these findings were suggestive of HCP, the authors noted an elevation of ALA that was higher than that of PBG and, furthermore, an increase of erythrocyte zinc protoporphyrin, suggesting an additional ALAD deficiency. Subsequent automated sequencing of both the CPOX and ALAD gene led to the detection of disease-causing mutations in each gene. Both genetic defects were confirmed by in vitro expression experiments, thereby for the first time unequivocally establishing the molecular basis in an individual with a dual porphyria consisting of a simultaneous CPOX and ALAD deficiency (33).
We strongly believe that the excellent molecular studies performed by the groups of Harraway and Akagi, respectively, point the way to the basic standards that should be fulfilled in the future before the diagnosis of a dual porphyria can be accepted. In our eyes, it should be mandatory that, from now on, all biochemical and enzymatic studies that suggest the simultaneous deficiency of two enzymes along the haeme biosynthetic pathway should always be complemented and confirmed by molecular genetic analysis of the corresponding genes to identify the disease-causing mutations underlying a particular variant of dual porphyria.