Andrew G. Roberts

Increased frequency of the haemochromatosis Cys282Tyr mutation in sporadic porphyria cutanea tarda.

BACKGROUND Sporadic porphyria cutanea tarda is a skin disease associated with hepatic siderosis. Depletion of iron stores by phlebotomy is curative. The role of haemochromatosis genes in determining susceptibility to this disorder is controversial. We have examined the frequency in sporadic porphyria cutanea tarda of mutations (Cys282Tyr, His63Asp) in a novel MHC class-I-like gene, one of which (Cys282Tyr) is believed to cause haemochromatosis. METHODS 41 patients with sporadic porphyria cutanea tarda, in whom the frequency of microsatellite alleles that define the ancestral haemochromatosis haplotype had previously been determined, and 101 healthy blood donors were studied for the presence of the Cys282Tyr and His63Asp mutations. We used restriction-enzyme digestion of PCR-amplified genomic DNA. FINDINGS The Cys282Tyr mutation occurred in 18 (44%) of patients compared with 11 (11%) of controls (relative risk 6.2, 95% CI 2.6-14.5, p = 0.00003). Seven (17%) patients, aged 48-79 years, were homozygotes. In 12 patients, the Cys282Tyr mutation was associated with markers of the HLA-A3-containing ancestral haemochromatosis haplotype. Ages at presentation were the same for those with or without the Cys282Tyr mutation. There was no difference in the frequency of the His63Asp mutation. INTERPRETATION Inheritance of one or more haemochromatosis genes is an important susceptibility factor for sporadic porphyria cutanea tarda. Some homozygotes for the Cys282Tyr mutation present late in life with porphyria cutanea tarda, indicating that not all homozygotes present clinically with haemochromatosis. The relation between this genotype and disease needs further investigation.

Variegate Porphyria in Western Europe: Identification of PPOX Gene Mutations in 104 Families, Extent of Allelic Heterogeneity, and Absence of Correlation between Phenotype and Type of Mutation

Variegate porphyria (VP) is a low-penetrance, autosomal dominant disorder characterized clinically by skin lesions and acute neurovisceral attacks that occur separately or together. It results from partial deficiency of protoporphyrinogen oxidase encoded by the PPOX gene. VP is relatively common in South Africa, where most patients have inherited the same mutation in the PPOX gene from a common ancestor, but few families from elsewhere have been studied. Here we describe the molecular basis and clinical features of 108 unrelated patients from France and the United Kingdom. Mutations in the PPOX gene were identified by a combination of screening (denaturing gradient gel electrophoresis, heteroduplex analysis, or denaturing high-performance liquid chromatography) and direct automated sequencing of amplified genomic DNA. A total of 60 novel and 6 previously reported mutations (25 missense, 24 frameshift, 10 splice site, and 7 nonsense) were identified in 104 (96%) of these unrelated patients, together with 3 previously unrecognized single-nucleotide polymorphisms. VP is less heterogeneous than other acute porphyrias; 5 mutations were present in 28 (26%) of the families, whereas 47 mutations were restricted to 1 family; only 2 mutations were found in both countries. The pattern of clinical presentation was identical to that reported from South Africa and was not influenced by type of mutation. Our results define the molecular genetics of VP in western Europe, demonstrate its allelic heterogeneity outside South Africa, and show that genotype is not a significant determinant of mode of presentation.

Genetics and pathogenesis of human uroporphyrinogen decarboxylase defects

Two types of human porphyria, porphyria cutanea tarda (PCT) and hepatoerythropoietic porphyria (HEP), result from partial deficiency of uroporphyrinogen decarboxylase (UROD). About 20% of patients with PCT have a 50% decrease in UROD concentration in all tissues that is inherited as an autosomal dominant trait with low penetrance (type II PCT). Both this condition and its postulated homozygous counterpart, HEP, show genetic heterogeneity. Identification of a form of familial PCT in which the activity and concentration of erythrocyte UROD is normal, as in type I or sporadic PCT, suggests than an autosomal gene, not necessarily at the UROD locus, may be important in determining the onset of type I PCT. Clinically overt PCT results from a liver-specific process that causes reversible inactivation of UROD and which may be iron dependent. The predisposition to develop PCT in response to common hepatotoxic agents and other acquired factors may be determined by interaction between genes that control the concentration of active UROD in cells and genes that facilitate the inactivation process.

Seasonal Palmar Keratoderma in Erythropoietic Protoporphyria Indicates Autosomal Recessive Inheritance

Erythropoietic protoporphyria (EPP) is an inherited disorder that results from partial deficiency of ferrochelatase (FECH). It is characterized clinically by acute photosensitivity and, in 2% of patients, liver disease. Inheritance is usually autosomal dominant with low penetrance but is recessive in about 4% of families. A cross-sectional study of 223 patients with EPP in the United Kingdom identified six individuals with palmar keratoderma. We now show that these and three additional patients, from six families, have an inherited subtype of EPP which is characterized by seasonal palmar keratoderma, relatively low erythrocyte protoporphyrin concentrations, and recessive inheritance. No patient had evidence of liver dysfunction; four patients had neurological abnormalities. Patients were hetero- or homoallelic for nine different FECH mutations; four of which were previously unreported. Prokaryotic expression predicted that FECH activities were 2.7-25% (mean 10.6%) of normal. Neither mutation type nor FECH activity provided an explanation for the unusual phenotype. Our findings show that palmar keratoderma is a clinical indicator of recessive EPP, identify a phenotype that occurs in 38% of reported families with recessive EPP that to our knowledge is previously unreported, and suggest that patients with this phenotype may carry a lower risk of liver disease than other patients with recessive EPP.

Heterogeneity of familial porphyria cutanea tarda.

The concentration of immunoreactive uroporphyrinogen decarboxylase has been measured in erythrocytes from 17 patients with porphyria cutanea tarda (PCT) from 10 families, from 74 of their relatives, and from 47 control subjects. The 10 families were divided into two groups according to their erythrocyte enzyme concentrations. Group A contained four families in which at least two subjects had overt PCT. All members of these families, including seven patients with overt PCT, had normal erythrocyte uroporphyrinogen decarboxylase concentrations and activities. Apart from their family history, patients in group A were clinically and biochemically indistinguishable from cases of type I (sporadic) PCT. Group B contained six families with the only previously described form of familial PCT (type II PCT) in which decreased erythrocyte uroporphyrinogen decarboxylase segregates as an autosomal dominant trait. These findings show that familial PCT is heterogeneous and suggest that inheritance contributes to the pathogenesis of at least some cases of type I PCT.

Prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria and porphyria cutanea tarda

SummaryThe prevalence of the 281 (Gly→Glu) mutation in hepatoerythropoietic porphyria (HEP) was investigated by the use of hybridization with a synthetic oligonucleotide probe. The mutation was found in HEP-affected members of two unrelated families from Spain, but was absent in two other patients from Italy and Portugal who also had HEP. Moreover, this mutation was not detected in 13 unrelated cases of familial (type II) porphyria cutanea tarda.

Purification and properties of uroporphyrinogen decarboxylase from human erythrocytes

Publisher Summary Uroporphyrinogen decarboxylase (UROD) is a cytosolic enzyme of the heme biosynthetic pathway. This chapter discusses the purification and properties of uroporphyrinogen decarboxylase from human erythrocytes. Erythrocytes are, therefore, a convenient source from which homogeneous preparations of UROD can be obtained in relatively good yield. The enzyme has also been purified to homogeneity from chicken erythrocytes, bovine liver, yeast, and Rhodobacter spheroides . All stages of the purification procedure are carried out at 4°. Dithiothreitol (DTT) is added to all buffers immediately before the use to a final concentration of 0.5 m M . Uroporphyrinogen decarboxylase activity is monitored at each stage of the purification procedure by measuring the conversion of pentacarboxylate porphyrinogen III to coproporphyrinogen III or by a modification in which porphyrins are not converted to their methyl esters but separated directly by reversed-phase high-performance liquid chromatography. For some preparations, UROD concentration is measured by electroimmunoassay, using a rabbit polyclonal antiserum to purified human erythrocyte UROD.

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.

De-novo mutation and sporadic presentation of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a low-penetrant autosomal dominant disorder characterised by life-threatening neurovisceral attacks, often precipitated by drugs. Prognosis is improved by presymptomatic diagnosis and counselling. We found that 29 of 103 (28%) unrelated patients presented as sporadic cases. One patient had a de-novo mutation. The same mutation has arisen de novo at least three times in 12% of British AIP families. About 3% (95% CI 1-8%) of all AIP index cases may have de-novo mutations. These findings emphasise the high frequency of sporadic presentation of AIP and indicate the limitations of presymptomatic diagnosis as an aid to management.

AMNIOTIC FLUID CHOLINESTERASE MEASUREMENT AS A RAPID METHOD FOR THE EXCLUSION OF FETAL NEURAL-TUBE DEFECTS

Amniotic fluid total cholinesterase (ChE) and acetylcholinesterase (AChE) activities have been measured in 404 pregnancies without fetal malformation and 79 pregnancies associated with open neural-tube defects (NTDs). Neither measurement, either alone or in combination, gave complete separation of the two groups. However, measurement of ChE can be used to assess the probability that a woman is carrying a fetus with an open NTD. Since ChE can be measured within 15 minutes and shows at least 70% of women selected for amniocentesis by serum alpha-fetoprotein screening to have a less than 1 in 100 chance of carrying a fetus with an open NTD, we suggest that this test may be used in an amniocentesis clinic both to reassure women with normal pregnancies and to select probable abnormalities for immediate further investigation.