Molecular epidemiology of erythropoietic protoporphyria in the U.K.

BACKGROUND: Erythropoietic protoporphyria (EPP) is a cutaneous porphyria caused by mutations in the ferrochelatase (FECH) or, less frequently, the delta-aminolaevulinate synthase 2 (ALAS2) gene. Predictive genetic counselling requires accurate molecular diagnosis and knowledge of patterns of inheritance. OBJECTIVES: To investigate the molecular epidemiology of EPP in the U.K. METHODS: DNA samples from 191 unrelated patients resident in the U.K. were analysed for mutations in the FECH and ALAS2 genes and for the FECH IVS3-48 dimorphism. RESULTS: Mutations were identified in 179 (94%) patients. Most (169; 94%) had a FECH mutation on one allele and were classified as having pseudodominant EPP (psdEPP); seven (4%) patients had FECH mutations on both alleles (autosomal recessive EPP) and three (2%) patients had ALAS2 mutations (X-linked dominant protoporphyria). The FECH IVS3-48C allele was strongly associated with psdEPP and with the absence of mutations at the FECH or ALAS2 loci. Fifty-six FECH mutations were identified, 19 being previously unreported. Missense mutations were predominant in autosomal recessive EPP (82%) but not in psdEPP (32%). One mutation (c.314 + 2T>G) was present in 41 (24%) of EPP families, most of whom appeared to be descended from a common ancestor resident in the north of England. CONCLUSIONS: These data define the prevalence and molecular epidemiology of each type of EPP in the U.K.

The molecular genetics of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is a syndrome in which accumulation of protoporphyrin IX in erythroid cells, plasma, skin and liver leads to acute photosensitivity and, in about 2% of patients, liver disease. More than 95% of unrelated patients have ferrochelatase (FECH) deficiency (MIM 177000) while about 2% have X-linked dominant protoporphyria (XLDPP) (MIM 300752) caused by gain-of-function mutations in the ALAS2 gene. Most FECH-deficient patients are compound heterozygotes for a hypomorphic allele (FECH IVS3-48C) and a deleterious FECH mutation that together lower FECH activity to around 30% of normal. The frequency of the IVS3-48C allele varies between populations, ranging from less than 1% to 45%. About 4% of unrelated FECH-deficient patients are compound heterozygotes or homozygotes for rare FECH mutations and have lower enzyme activities. Acquired somatic mutation of FECH secondary to myeloid disease may rarely cause EPP. The risk of liver disease is increased in XLDPP and in FECH-deficient patients who are hetero- or homoallelic for rare FECH mutations. Inherited FECH-deficient EPP is an autosomal recessive disorder with some families showing pseudodominant inheritance; the proportion of such families being determined by the population frequency of the IVS3-48C allele.

Serum 25-hydroxyvitamin D in erythropoietic protoporphyria.

BACKGROUND: Vitamin D, produced by the action of sunlight on skin, is an important hormone for calcium homeostasis and has been implicated as tumour-protective agent. Some previous studies of photosensitive patients who actively avoid sunlight have failed to show convincing evidence of vitamin D insufficiency. OBJECTIVES: The aim of this study was to characterize the vitamin D status of a large cohort of patients with erythropoietic protoporphyria (EPP). METHODS: U.K. patients with EPP were recruited prospectively and seen locally by a single study investigator. A blood sample was taken for vitamin D assay. All blood analyses were performed in the same laboratory. RESULTS: A cohort of 201 patients with known EPP was seen over a 7-month period between January and July. Thirty-four patients (17%) were deficient in vitamin D and 126 (63%) had insufficient vitamin D. Both insufficiency and deficiency were significantly associated with the total erythrocyte protoporphyrin concentration and inversely with the time in minutes to the onset of symptoms following sunlight exposure. CONCLUSIONS: This is the first report of significant levels of vitamin D deficiency and insufficiency in a large cohort of patients with a photodermatosis. Such individuals are at risk of associated adverse events. In future, clinicians should consider monitoring 25-hydroxyvitamin D levels and instigating oral supplementation or dietary advice if appropriate.

Late presentation of erythropoietic protoporphyria: case report and genetic analysis of family members.

Erythropoietic protoporphyria (EPP) is an inherited disorder of haem biosynthesis caused by decreased activity of the enzyme ferrochelatase (FECH), which catalyses the insertion of iron into protoporphyrin, the last step in haem biosynthesis. Development of clinically overt EPP usually requires inheritance of a severe FECH mutation trans to a low-expression FECH variant (FECH IVS3-48C), which is present in 13% of the U.K. population. Reduced FECH activity leads to accumulation of protoporphyrin in various tissues. An excess amount of free protoporphyrin in the skin causes photosensitivity. EPP usually presents in early childhood or infancy, with painful burning and pruritus within minutes of light exposure. Onset of symptoms in adults is rare and often associated with acquired somatic mutation of the FECH gene secondary to haematological malignancy. Here we describe a patient with EPP, in whom the presenting clinical symptom, night-time itch, did not appear until middle age and who had an asymptomatic sister with the same FECH genotype.

Erythropoietic protoporphyria in the U.K.: clinical features and effect on quality of life.

BACKGROUND: Erythropoietic protoporphyria (EPP) is a rare inherited photodermatosis that causes lifelong painful photosensitivity. Neither its full clinical spectrum nor its impact on quality of life (QoL) has been investigated in a large cohort of patients. OBJECTIVES: To document the clinical features of EPP and its impact on QoL in a high proportion of all patients with EPP resident in the U.K. METHODS: Patients with EPP were identified from U.K. clinical databases and assessed by the same clinical investigator over a 7-month period using a standardized proforma and validated adult (Dermatology Life Quality Index, DLQI) and children's (Children's Dermatology Life Quality Index, CDLQI) QoL questionnaires. RESULTS: Three hundred and eighty-nine living patients with EPP were identified, of whom 223 [114 females, 109 males; median age 34 years (range: 5-87), from 193 families] were investigated. Total erythrocyte porphyrin (TEP) was higher in males (median: 25.3 micromol L1) than females (median: 19.3 micromol L1). The median ages at onset and diagnosis were 1 and 12 years, respectively. Median times for onset of symptoms after sun exposure, onset of signs (oedema, erythema) and resolution of symptoms were 20 min, 6 h and 3 days, respectively. Most patients reported absence of protection by glass (92%), priming (85%), exacerbation by wind (68%), no family history of photosensitivity (56%), no symptoms during winter (56%) and had chronic skin lesions (79%). Symptoms changed little with age but improved during pregnancy in 47% of gravid women. Most patients used protective clothing and a sunscreen; 28% were taking beta-carotene and a further 56% had taken it; 29% were not under regular medical care. Two patients (1%) had liver failure and 8% reported gallstone disease. QoL was markedly impaired, with scores similar to those in severe dermatological disease (mean DLQI score 14.0, n = 176; mean CDLQI score 12.8, n = 44), indicating a large effect on patients' lives. DLQI scores correlated weakly with TEP (rs = 0.228; P = 0.002) and time to onset of symptoms (rs = -0.233; P = 0.002) but not with age at onset. CONCLUSIONS: EPP is a persistent, severely painful, socially disabling disease with a marked impact on QoL. Its diagnosis is often overlooked. None of TEP, age at onset nor time to onset of symptoms is a useful predictor of impaired QoL in individual patients.

Molecular mechanisms of dominant expression in porphyria.

Partial deficiency of enzymes in the haem synthetic pathway gives rise to a group of seven inherited metabolic disorders, the porphyrias. Each deficiency is associated with a characteristic increase in haem precursors that correlates with the symptoms associated with individual porphyrias and allows accurate diagnosis. Two types of clinical presentation occur separately or in combination; acute life-threatening neurovisceral attacks and/or cutaneous symptoms. Five of the porphyrias are low-penetrance autosomal dominant conditions in which clinical expression results from additional factors that act by increasing demand for haem or by causing an additional decrease in enzyme activity or by a combination of these effects. These include both genetic and environmental factors. In familial porphyria cutanea tarda (PCTF), environmental factors that include alcohol, exogenous oestrogens and hepatotropic viruses result in inhibition of hepatic enzyme activity via a mechanism that involves excess iron accumulation. In erythropoietic protoporphyria (EPP), co-inheritance of a functional polymorphism in trans to a null ferrochelatase allele accounts for most clinically overt cases. In the autosomal dominant acute hepatic porphyrias (acute intermittent porphyria, variegate porphyria, hereditary coproporphyria), acute neurovisceral attacks occur in a minority of those who inherit one of these disorders. Although various exogenous (e.g. drugs, alcohol) and endogenous factors (e.g. hormones) have been identified as provoking acute attacks, these do not provide a full explanation for the low penetrance of these disorders. It seems probable that genetic background influences susceptibility to acute attacks, but the genes that are involved have not yet been identified.

Hepatoerythropoietic porphyria: a missense mutation in the UROD gene is associated with mild disease and an unusual porphyrin excretion pattern.

Hepatoerythropoietic porphyria (HEP) is an uncommon inherited cutaneous porphyria, related to porphyria cutanea tarda, that results from severe uroporphyrinogen decarboxylase (UROD) deficiency. It is characterized clinically by the onset in early childhood of severe lesions on sun-exposed skin. We describe a man aged 38 years with an unusually mild form of the disease that started in his early teens. Our data confirm that homozygosity for the F46L mutation in the UROD gene causes a mild form of HEP and show that this genotype may be associated with a unique urinary porphyrin excretion pattern in which pentacarboxylic porphyrin predominates.

Ferrochelatase gene polymorphism analysis for accurate genetic counselling in erythropoietic protoporphyria.

It has recently been shown that most cases of clinically overt erythropoietic protoporphyria (EPP) result from coinheritance of a mutated ferrochelatase gene and a commonly occurring low-expression normal variant allele. The identification of two polymorphic variant sequences associated with this low-expression allele now enables improved predictive counselling for couples where one partner has EPP. We describe a patient and his spouse in whom we have used such genetic analysis to provide an accurate estimate of the chance that their future offspring may suffer from EPP.

Management of acute and cutaneous porphyrias.

The porphyrias comprise a group of disorders of the haem biosynthesis pathway that can present with acute neurovisceral symptoms, skin lesions or both. Acute porphyrias present with severe abdominal pain, confusion and seizures which may be life-threatening. Specific treatment with haem preparations should be instituted as soon as possible following confirmation of increased excretion of porphobilinogen in the urine. Supportive treatment includes opiate analgesia, monitoring for and treating complications such as hypertension and hyponatraemia. Follow-up should include counselling on lifestyle modification involving avoidance of alcohol, smoking and known porphyrogenic drugs and diet. Identification and counselling of at risk relatives is essential. The cutaneous porphyrias result from porphyrin-induced photosensitivity and can present with either acute photosensitivity or skin fragility and blisters. All cutaneous porphyrias can be alleviated by avoidance of sunlight. Treatment of erythropoietic protoporphyria involves administering large doses of beta-carotene, which may improve tolerance to sunlight. Porphyria cutanea tarda can be effectively treated by phlebotomy or low dose chloroquine. Congenital erythropoietic porphyria is a rare, early onset, severe, photomutilating condition for which bone marrow transplantation has been shown to be successful.

Parent-adolescent reciprocity in negative affect and its relation to early adult social development.

The purposes of this 9-year, prospective longitudinal study were (a) to investigate hypothesized reciprocal growth in negative emotions between parents and adolescents and (b) to examine the influence of this reciprocal process on the development of social relationships during early adulthood. The results showed that both parents' and adolescents' initial levels of negative emotion toward each other predicted the rate of growth and rate of change in growth of expressed negative affect. In addition, the analyses indicated that an adolescent's enmeshment in reciprocal negativity in the family of origin carried over into early adult social relationships. The findings demonstrate the reciprocal nature of negative affect in interactions between parents and adolescents and suggest that family experience with this interactional style may have an adverse influence on the development of early adult social relationships.

Treatment of acute porphyria.

Haem preparations are now available for the specific treatment of attacks of acute porphyria. This review focuses on their use in this uncommon but life-threatening medical emergency.

ACP Best Practice No 165: front line tests for the investigation of suspected porphyria.

The porphyrias are uncommon disorders of haem biosynthesis and their effective management requires prompt and accurate diagnosis. This article describes methods for the determination of urinary porphobilinogen, urinary and faecal total porphyrins, and total porphyrins in erythrocytes and plasma that are suitable for use in non-specialist laboratories. The selection and interpretation of these methods, and the indications for further, more specialised, investigation are discussed.

Pubertal transition, stressful life events, and the emergence of gender differences in adolescent depressive symptoms.

This study examined the role of both pubertal and social transitions in the emergence of gender differences in depressive symptoms during adolescence. This study generated the following findings: (a) Gender differences in depressive symptoms emerged during 8th grade and remained significant through 12th grade. (b) Pubertal status in 7th grade was related to adolescent depressive symptoms over time. (c) Early-maturing girls represented the group with the highest rate of depressive symptoms. (d) Depressive symptoms measured in 7th grade predicted subsequent symptom levels throughout the secondary school years. (e) Recent stressful life events were associated with increased depressive symptoms. (f) Early-maturing girls with higher levels of initial symptoms and more recent stressful life events were most likely to be depressed subsequently. The findings demonstrate the importance of the interaction between the pubertal transition and psychosocial factors in increasing adolescent vulnerability to depressive experiences.

Characterization of mutations in the CPO gene in British patients demonstrates absence of genotype-phenotype correlation and identifies relationship between hereditary coproporphyria and harderoporphyria.

Hereditary coproporphyria (HCP) is the least common of the autosomal dominant acute hepatic porphyrias. It results from mutations in the CPO gene that encodes the mitochondrial enzyme, coproporphyrinogen oxidase. A few patients have also been reported who are homoallellic or heteroallelic for CPO mutations and are clinically distinct from those with HCP. In such patients the presence of a specific mutation (K404E) on one or both alleles produces a neonatal hemolytic anemia that is known as "harderoporphyria"; mutations on both alleles elsewhere in the gene give rise to the "homozygous" variant of HCP. The molecular relationship between these disorders and HCP has not been defined. We describe the molecular investigation and clinical features of 17 unrelated British patients with HCP. Ten novel and four previously reported CPO mutations, together with three previously unrecognized single-nucleotide polymorphisms, were identified in 15 of the 17 patients. HCP is more heterogeneous than other acute porphyrias, with all but one mutation being restricted to a single family, with a predominance of missense mutations (10 missense, 2 nonsense, 1 frameshift, and 1 splice site). Of the four known mutations, one (R331W) has previously been reported to cause disease only in homozygotes. Heterologous expression of another mutation (R401W) demonstrated functional properties similar to those of the K404E harderoporphyria mutation. In all patients, clinical presentation was uniform, in spite of the wide range (1%-64%) of residual coproporphyrinogen oxidase activity, as determined by heterologous expression. Our findings add substantially to knowledge of the molecular epidemiology of HCP, show that single copies of CPO mutations that are known or predicted to cause "homozygous" HCP or harderoporphyria can produce typical HCP in adults, and demonstrate that the severity of the phenotype does not correlate with the degree of inactivation by mutation of coproporphyrinogen oxidase.

Homozygous variegate porphyria: a compound heterozygote with novel mutations in the protoporphyrinogen oxidase gene.

Homozygous variegate porphyria results from mutations in both alleles of the protoporphyrinogen oxidase (PPOX) gene. Our patient, a 36-year-old woman, has severe cutaneous manifestations. Her clinical and biochemical features are similar to the few other reported cases, including onset before 18 months of age, photosensitivity, absence of acute porphyric attacks, and elevated erythrocyte protoporphyrin. Mutation analysis of the PPOX gene revealed an in-frame 12 bp insert (c. 657-658 ins AAGGCCAGCGCC) encoding lysine-alanine-serine-alanine (KASA), and a G to A transition at the splice donor site of exon 11 (IVS 11-1 G-->A). Neither of these mutations has been reported previously. Our patient's mother has the splice site mutation and has had acute porphyric episodes. A maternal first cousin has the same mutation but no clinical manifestations. The medical and family history of our patient's father is uncertain.

Alternative splicing and tissue-specific transcription of human and rodent ubiquitous 5-aminolevulinate synthase (ALAS1) genes.

The rate of haem synthesis in non-erythroid mammalian tissues is controlled by the ubiquitous isoform of 5-aminolevulinate synthase (ALAS1). In order to explore the regulation of mammalian ALAS1 genes, we have investigated the transcription of the human and rat genes. The 17 kb human gene differs from the rat gene in containing an additional untranslated exon that is alternatively spliced to produce a longer, minor mRNA transcript. Relative amounts of the two transcripts were similar in all tissues examined. Analysis of mRNA transcripts in human and rat tissues revealed tissue-specific differences in the use of transcription start sites by closely similar core promoters. In brain, initiation was from sites within and upstream from the TATA box, including an initiator-like element. In liver, initiation was TATA-driven from a single downstream site that appeared to be used exclusively for induction by drugs. Intermediate patterns were observed in other tissues and cell lines. Mutation of the TATA box did not impair transcription in transfected HeLa cells but activated upstream start sites, recapitulating the brain pattern. Our findings indicate that the conformation of the core ALAS1 promoter that directs assembly of the transcription pre-initiation complex may vary between tissues and have implications for understanding the tissue-specific regulated expression of this gene.

Late-onset erythropoietic porphyria caused by a chromosome 18q deletion in erythroid cells.

The erythropoietic porphyrias, erythropoietic protoporphyria and congenital erythropoietic porphyria, result from germline mutations in the ferrochelatase gene and uroporphyrinogen III synthase gene, respectively. Both conditions normally present in childhood but rare cases with onset past the age of 40 y have been reported. Here we show that late-onset erythropoietic protoporphyria can be caused by deletion of the ferrochelatase gene in hematopoietic cells with clonal expansion as part of the myelodysplastic process. This is the first direct demonstration of porphyria produced by an acquired molecular defect restricted to one tissue. Some other cases of late-onset erythropoietic porphyria may be explained by a similar mechanism.

Non-erythroid form of acute intermittent porphyria caused by promoter and frameshift mutations distant from the coding sequence of exon 1 of the HMBS gene.

Acute intermittent porphyria (AIP) is a low-penetrant, autosomal dominant disorder caused by mutations in the HMBS gene. The gene is transcribed from two promoters to produce ubiquitous and erythroid isoforms of porphobilinogen deaminase, which differ only at their NH2 ends. In the classical form of AIP, both isoforms are deficient, but about 5% of families have the non-erythroid variant in which only the ubiquitous isoform is affected. Previously identified mutations in this variant have been within or close to the coding region of exon 1 of the HMBS gene, the only exon that is expressed solely in the ubiquitous isoform. Here, we describe mutations in the ubiquitous promoter (-154delG) and in exon 3 (41delA) that cause the non-erythroid variant. Reporter gene and electrophoretic mobility shift assays show that the G nucleotide at position -154, the most 5' of several transcription-initiation sites in the ubiquitous HMBS promoter, which lies immediately 3' to a transcription-factor IIB binding motif, is essential for normal transcription. The frameshift mutation in exon 3 introduces a stop codon into mRNA for the ubiquitous isoform only. Our investigations identify two new mechanisms for production of the non-erythroid variant of AIP and demonstrate that mutational analysis for diagnosis of this variant needs to include wider regions of the HMBS gene than indicated by previous reports. Furthermore, they show that deletion of one of several transcription initiation sites in the promoter of a housekeeping gene that lacks both TATA and initiator elements can produce disease.