Theodore Woodward Award. Pathogenesis of biochemical abnormalities in protoporphyria.

In summary, FC gene mutations in patients with protoporphyric liver disease typically cause major structural alterations in the FC protein. However, the gene mutations by themselves do not satisfactorily account for the severe phenotype, as the same mutations are found in asymptomatic family members, and similar mutations are found in patients who do not develop liver disease. Thus there may be unidentified factors in the FC gene locus, or factors outside the locus, which are also important in determining the degree of protoporphyrin accumulation that occurs in an individual patient, hence, the potential for developing significant liver disease. Further studies are needed to clarify this possibility and identify those factors.

Erythropoietic protoporphyria: identification of novel mutations in the ferrochelatase gene and comparison of biochemical markers versus molecular analysis as diagnostic strategies.

BACKGROUND: Erythropoietic protoporphyria (EPP) results from an inherited deficiency of the last enzyme of the heme biosynthetic pathway, ferrochelatase (FC). EPP is usually inherited in an autosomal dominant fashion, and the mutations in the FC gene on chromosome 18q21.3 detected in EPP patients are heterogeneous. METHODS: In this study, we screened the FC gene for mutations in 12 patients from 10 unrelated families with EPP and their family members using heteroduplex analysis, automated sequencing, and restriction enzyme digestion. RESULTS: We detected 8 different mutations in these patients, including 1 missense mutation, 5 frameshift mutations, and 2 splice site mutations, 6 of which are previously undescribed. CONCLUSIONS: We have established the molecular basis of EPP in 10 unrelated families, thereby providing further evidence for the heterogeneity in this disorder. Importantly, molecular diagnosis allowed revisions in the status of several clinically unaffected silent mutation carriers within the families. We compare the value of genetic research strategies with the combination of biochemical data and clinical phenotype as diagnostic tools to confirm a putative diagnosis in EPP.

Mutations in the translation initiation codon of the protoporphyrinogen oxidase gene underlie variegate porphyria.

Variegate porphyria (VP), one of the acute hepatic porphyrias, is characterized by a reduced catalytic activity of protoporphyrinogen oxidase (PPO), the penultimate enzyme in the porphyrin-haem biosynthetic pathway. VP has been linked to the PPO gene on chromosome 1q22-23, and several mutations underlying this disorder have been described recently. In this study, we identified two different missense mutations in the translation initiation codon of the PPO gene in two unrelated patients with VP. Mutation analysis was carried out using PCR, heteroduplex analysis, automated sequencing, and restriction enzyme digestion. In the first patient, the results revealed an A-to-T transversion (ATG --> TTG), resulting in the substitution of methionine by leucine (M1L). The mutation detected in the second patient was a T-to-C transition (ATG --> ACG), leading to the conversion of methionine to threonine (M1T). These mutations abolish the initiation of translation at the normal site, and consequently, translation of an abnormal messenger RNA (mRNA) would result in the synthesis of a truncated PPO protein lacking the amino terminus.

Haplotype analysis of families with erythropoietic protoporphyria and novel mutations of the ferrochelatase gene.

Ferrochelatase, the enzyme that catalyzes the terminal step in the heme biosynthetic pathway, is the site of the defect in the human inherited disease erythropoietic protoporphyria. Molecular genetic studies have shown that the majority of erythropoietic protoporphyria cases are transmitted in dominant fashion and that mutations underlying erythropoietic protoporphyria are heterogeneous. We performed haplotype analysis of American families that shared recurrent ferrochelatase gene mutations yet had forbearers from several European countries. This was to gain insight into whether these mutations represent mutational hotspots at the ferrochelatase gene, or propagation of ancestral alleles bearing the mutations. Two recurrent mutations were found to occur on distinctive chromosome 18 haplotypes, consistent with being hotspot mutations. On the other hand, we found three sets of two unrelated families that shared the same haplotypes bearing these mutations, which could reflect geographic dispersion of ancestral mutant alleles. In addition, we report novel mutations associated with erythropoietic protoporphyria: g(+ 1)-->t transversion of the exon 4 donor site, g(+ 1)-->a transition of the exon 6 donor site, and t(+ 2)-->a substitution at the exon 9 donor site; these mutations are predicted to cause splicing defects of the associated exons. We also identified a g(+ 5)-->a transition of the exon 1 donor site in four unrelated families with erythropoietic protoporphyria, and a G(- 1)-->A substitution at the exon 9 donor site in an additional family. The probability that these sequence changes are normal polymorphisms was virtually excluded (p < 0.0001) by their absence in 120 ferrochelatase alleles from 30 normal subjects and 30 individuals with manifested erythropoietic protoporphyria with or without a known mutation.

C73R is a hotspot mutation in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) results from profoundly deficient activity of the fourth enzyme of the haeme biosynthetic pathway, uroporphyrinogen III synthase (UROIIIS). CEP is a rare, recessively inherited disorder, and mutations in the UROIIIS gene detected in CEP patients are heterogeneous. The notable exception to this rule is a single missense mutation, designated C73R, which represents over 40% of all mutant UROIIIS alleles. In this study, we investigated three separate families with CEP from different ethnic backgrounds. We performed haplotype analysis using two microsatellite markers that closely flank the UROIIIS gene on chromosome 10q24, spanning a region of 4 cM on the GB4 linkage panel. Haplotype analysis revealed the occurrence of C73R on different haplotypes in four out of four disease chromosomes studied. The results are consistent with the hypothesis that C73R is a hotspot mutation for CEP, and does not represent wide dispersion of a single ancestral mutant C73R allele.

The genetic basis of "Scarsdale Gourmet Diet" variegate porphyria: a missense mutation in the protoporphyrinogen oxidase gene.

The porphyrias are disorders of porphyrin or porphyrin-precursor metabolism that result from inherited or acquired aberrations in the control of the porphyrin-heme biosynthetic pathway. Variegate porphyria (VP), one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of protoporphyrinogen oxidase (PPO), and recently, mutations in the PPO gene on chromosome 1q22-23 have been described. Our purpose was to identify the underlying genetic lesion in a severely affected patient with VP and to detect the silent mutation carriers in her family. The disease in this patient was precipitated by carbohydrate restriction as outlined in the "Scarsdale Gourmet Diet". Our mutation detection and confirmation strategy included PCR, automated sequencing, and restriction enzyme digestion. We identified a missense mutation in the patient and five family members. The mutation consisted of a previously unreported C-to-T transition in exon 5 of the PPO gene, resulting in the substitution of arginine by cysteine, designated R152C. This arginine residue is evolutionarily highly conserved in humans, mice, bacteria, yeast, and plants, indicating the importance of this residue in PPO. Our study established that a missense mutation in the PPO gene was the underlying mutation in this patient with VP and explained the occurrence of the phenotype in this family.

Porphyria cutanea tarda, hepatitis C, and HFE gene mutations in North America.

In some, but not all countries, porphyria cutanea tarda (PCT) has been associated with chronic infection with the hepatitis C virus (HCV). Recently, PCT has also been associated with mutations in the HFE gene that are associated with HLA-linked hereditary hemochromatosis. Until now, few studies of these associations have been reported from North America. The aims of this study were: 1) to assess the prevalence of HCV infection and HFE mutations in North American patients with PCT; 2) to compare demographic and laboratory features between those who are HCV-positive and HCV-negative; and 3) to study urinary porphyrin excretions in American HCV-positive patients without clinically manifest PCT. Clinical and laboratory data, including tests for HCV and urinary porphyrins, were collected from 70 unselected patients with typical PCT. Urinary porphyrins were also measured in 110 non-PCT patients with chronic hepatitis C. Mutational analyses of the HFE gene were performed in 26 PCT patients. Thirty-nine of 70 (56%) of the PCT patients had evidence of HCV infection. Thirty-two of 39 PCT patients with HCV were men, all of whom used alcohol. In contrast, 22 of 31 PCT patients without HCV infection were women, 12 of whom had taken estrogens. The HCV-positive group was more likely to have used illicit intravenous drugs (45% vs. 0%; P = 0.01), to have had several (>4) sex partners (48% vs. 13%; P = 0.005), and less likely to have no known risk factors for HCV infection (33% vs. 78%; P = 0.004). Total urinary porphyrin excretion was the same in the two groups, but those with HCV infection had a significantly lower percentage of uroporphyrin and higher percentages of hepta-and hexa-carboxy porphyrins in urine. Sixteen of 110 (15%) HCV-positive subjects without PCT had increased urinary porphyrins, but, unlike PCT, these were mainly coproporphyrin. Forty-two percent of PCT patients carried the C282Y mutation of HFE (15% homozygous), and another 31% carried the H63D mutation (8% homozygous). Thus, 73% of PCT patients had one of these mutations. The prevalence of HCV infection (56%) and mutations in the HFE gene (73%) are high among North American patients with PCT. Alcohol and estrogen use are important additional risk factors. All PCT patients should be tested for HCV infection and for HFE gene mutations. Although HCV infection is a trigger for PCT, preclinical PCT is rare in chronic HCV hepatitis C in the United States.

Molecular basis of variegate porphyria: a missense mutation in the protoporphyrinogen oxidase gene.

Variegate porphyria (VP) is an autosomal dominant disorder characterised by a partial defect in the activity of protoporphyrinogen oxidase (PPO), and has recently been genetically linked to the PPO gene on chromosome 1q22-23 (Z=6.62). In this study, we identified a mutation in the PPO gene in a patient with VP and two unaffected family members. The mutation consisted of a previously unreported T to C transition in exon 13 of the PPO gene, resulting in the substitution of a polar serine by a non-polar proline (S450P). This serine residue is evolutionarily highly conserved in man, mouse, and Bacillus subtilis, attesting to the importance of this residue. Interestingly, the gene for Gardner's syndrome (FAP) also segregates in this family, independently of the VP mutation. Gardner's syndrome or familial adenomatous polyposis (FAP) is also an autosomal dominantly inherited genodermatosis, and typically presents with colorectal cancer in early adult life secondary to extensive adenomatous polyps of the colon. The specific gene on chromosome 5 that is the site of the mutation in this disorder is known as APC (adenomatous polyposis coli), and the gene has been genetically linked to the region of 5q22.

Erythropoietic protoporphyria: four novel frameshift mutations in the ferrochelatase gene.

Human erythropoietic protoporphyria is an inherited disorder of the heme metabolic pathway caused by defects in the gene for ferrochelatase, the terminal enzyme of the pathway that catalyzes chelation of ferrous iron into protoporphyrin IX to form heme. Mutation analysis was performed for families with erythropoietic protoporphyria and four novel frameshift mutations were identified. Two of the mutations, 205insA and 215insT in exon 3 of the ferrochelatase gene, are single bp insertions. The other two, 400delA in exon 4 and 678delG in exon 6, are single bp deletions. All of the mutations result in premature termination codons downstream shortly after the mutation sites, and in one case the premature termination codon caused by 400delA was also shown to reduce mRNA level via nonsense-mediated mRNA decay.

Molecular basis of variegate porphyria: a de novo insertion mutation in the protoporphyrinogen oxidase gene.

The porphyrias are disorders that result from the inherited or acquired dysregulation of one of the eight enzymes in the heme biosynthetic pathway. Variegate porphyria (VP) is characterized by deficiencies in protoporphyrinogen oxidase (PPO) and has recently been genetically linked (Z = 6.62) to the PPO gene on chromosome 1q21. In this study, we have identified two sequence variants in the PPO gene in a family with VP. The first is a neutral polymorphism at the -47 position of intron 2; this polymorphism is present in the general population and is unlikely to underlie the VP phenotype. The second is a mutation in the PPO gene in a patient with VP; the mutation consists of an apparently de novo 2-bp insertion in exon 3 of PPO and results in a frameshift and downstream premature termination codon. These data establish that a frameshift mutation in PPO is the underlying mutation in this patient with VP and explain the sporadic occurrence of the phenotype in this family.

Human protoporphyria: reduced cutaneous photosensitivity and lower erythrocyte porphyrin levels during pregnancy.

BACKGROUND: Women with erythropoietic protoporphyria (EPP) have reported increased sunlight tolerance during pregnancy. Review of clinical information in an existing database for an EPP population study found five women who had six pregnancies while enrolled. All had experienced attenuated photosensitivity during gestation. OBJECTIVE: Our purpose was to gain insight into whether altered porphyrin metabolism during pregnancy might explain this phenomenon. METHODS: Erythrocyte protoporphyrin levels obtained for these women during six periods of gestation were compared with data accumulated over the course of several years during nongestational periods. RESULTS: Erythrocyte porphyrin levels were lower during pregnancy. The differences of the means for values obtained during nongestational periods versus values obtained during pregnancy for each woman were found to reach or approach significance (p < 0.05) by a paired t test when analyzed without regard for seasonality (p = 0.042) or when adjusted for possible seasonal effects of sunlight on erythrocyte porphyrin levels by separation into data sets for low sunlight months (October-April) (p = 0.039) or high sunlight months (May-September) (p = 0.057). CONCLUSION: These observations suggest that a beneficial physiologic effect of pregnancy in patients with EPP may be a lower circulating erythrocyte protoporphyrin burden that leads to reduced photosensitivity.

Congenital erythropoietic porphyria: clinical, biochemical, and enzymatic profile of a severely affected infant.

Blistering of light-exposed skin, pink-stained fluorescing diapers, and fluorescing peripheral erythrocytes led to diagnosis of congenital porphyria in an infant born to consanguineous parents. Although massive coproporphyrinuria and coproporphyrinemia initially suggested a coproporphyrinogen oxidase deficiency disorder, excess porphyrins were chiefly of the isomer I series, implicating a uroporphyrinogen III synthase defect. Congenital erythropoietic porphyria was confirmed by demonstration of a profound defect in the activity of the infant's uroporphyrinogen III synthase (4% of the mean value for nine normal controls) and in both parents at approximately 50% of the mean normal activity. Coinheritance of gene defects for either hereditary coproporphyria or erythropoietic protoporphyria in addition to those for congenital erythropoietic porphyria was excluded by demonstrating normal activities of both coproporphyrinogen oxidase and ferrochelatase in the infant. The complicated perinatal and postnatal clinical course and biochemical and enzyme assay data for the infant and his parents are described.

Systematic screening for RNA with skipped exons--splicing mutations of the ferrochelatase gene.

A systematic method was designed to screen a large population of patients with erythropoietic protoporphyria (EPP) for aberrant ferrochelatase RNA with skipped exons. The method utilizes the new junction sequence created by exon skipping as the probe to detect such RNA species. In 7 of 17 EPP families, an aberrant ferrochelatase RNA with one exon missing was observed. Two previously unreported splicing mutations were also identified in 2 EPP families. One was a G >> T transversion at the +1 position of the acceptor site of intron 8, causing exon 9 to be skipped during RNA splicing. Both the patient and her father were found to be heterozygous for this mutation. In another family, an A >> G transition at the +3 position of the donor site of intron 10 was identified, associated with exon 10 skipping during RNA splicing. Both the patient and her father were heterozygous for this mutation.

Human protoporphyria: genetic heterogeneity at the ferrochelatase locus.

Inherited deficiency of ferrochelatase results in erythropoietic protoporphyria (EPP). Genetic heterogeneity at the locus for human ferrochelatase was investigated. Analysis of genomic DNA of patients with EPP and of control subjects by restriction endonuclease techniques using ten different enzymes detected polymorphisms only at sites recognized by EcoRI, HincII, PstI and TaqI. None of these polymorphisms alone was specific for expression of the disease since each was observed in control subjects as well. Three of these polymorphisms (at EcoRI, HincII and PstI sites) were always associated, indicating linkage. These and other studies demonstrate that the ferrochelatase gene is markedly heterogeneous. It is not yet clear whether some of the mutations associated with these polymorphisms contribute to expression of EPP.

A novel splicing mutation in the ferrochelatase gene responsible for erythropoietic protoporphyria.

An aberrant ferrochelatase mRNA lacking exon 7 was found in a patient with erythropoietic protoporphyria (EPP). The exon 7 skipping appears to result from a G >> A transition at position +5 of the donor site of intron 7 of the ferrochelatase gene. The patient is heterozygous for the mutation. Since the patient's paternal half-brother (not available for testing) also has clinically obvious EPP, their father appeared to be the source of the mutant allele. The father was in fact found to be a carrier of the same mutation and his ferrochelatase activity was 35% of normal; however, he is asymptomatic, with only a slightly elevated erythrocyte protoporphyrin level. These findings confirm that the observed mutation is responsible for the defect. The variability in clinical expression of EPP probably reflects the great heterogeneity of the ferrochelatase gene defects and the contribution of additional exogenous and endogenous inducers of latent disease.

Cutaneous porphyrialike photosensitivity after liver transplantation.

BACKGROUND: Liver disease and cholestasis are often associated with abnormal coproporphyrin levels, but photosensitivity eruptions do not usually occur with these conditions. We present a case of a cutaneous porphyrialike photosensitivity in a liver transplant patient with only mildly elevated coproporphyrin levels. OBSERVATIONS: A 5-year-old girl developed a cutaneous porphyria cutanea tarda-like photosensitivity after liver transplantation for congenital biliary atresia. Her cutaneous eruption consisted of fluid-filled vesicles and crusted erosions involving her face and arms. These lesions eventually healed with atrophic scarring and milia. The patient was in a state of chronic hepatic rejection, but her serum and 24-hour urine specimens contained only mildly elevated levels of coproporphyrin. Results of histopathologic examination and direct immunofluorescence of a skin biopsy specimen resembled those of porphyria. CONCLUSIONS: Although clinically porphyria cutanea tarda was suspected, her porphyrin profile was not consistent with that diagnosis. Liver transplantation has become relatively common, but porphyria cutanea tarda-like clinical and histopathologic findings associated with coproporphyrinemia in a liver transplant patient have not been reported to date.