Comprehensive cytochrome P450 CYP1A2 gene analysis in French caucasian patients with familial and sporadic porphyria cutanea tarda.

BACKGROUND: Porphyria cutanea tarda (PCT), the most frequent type of porphyria, results from decreased uroporphyrinogen decarboxylase (UROD) activity. Two forms of PCT have been described: a familial form (fPCT) characterized by the inherited decrease of UROD activity in all tissues and a sporadic form (sPCT) characterized by decreased UROD activity in the liver. Cytochrome P450 CYP1A2 plays a major role in triggering experimental uroporphyria in rodents. It has been suggested that the highly inducible -163A/A genotype of the CYP1A2 gene could confer a heightened risk of PCT in patients. OBJECTIVES: To examine the impact of CYP1A2 polymorphisms on the clinical course of PCT. METHODS: We performed an extensive CYP1A2 gene analysis in 96 (48 fPCT and 48 sPCT) unrelated French caucasian patients with PCT and in 99 healthy volunteers of similar ethnic origin. Results We did not observe any difference in CYP1A2 allele distribution, including a novel and rare CYP1A2 c.1063C>T (p.R355W) single nucleotide polymorphism. In addition, we compared the frequency of the -163A highly inducible allele both in patients with symptomatic fPCT (n = 48) and in asymptomatic UROD gene mutations carrier relatives (n=54). This variant was not over-represented in patients with PCT vs. either healthy volunteers or asymptomatic UROD gene mutation carriers. CONCLUSIONS: The CYP1A2 genotype does not appear to be a major susceptibility factor in the development of fPCT or sPCT in the French population.

Loss of heterozygosity on 10q and mutational status of PTEN and BMPR1A in colorectal primary tumours and metastases.

We investigated the possible role of chromosome 10q losses in colorectal cancer metastasis by carrying out an allelic imbalance study on a series of microsatellite instability-negative (MSI-) primary tumours (n=32) and metastases (n=36) from 49 patients. Our results demonstrate that 10q allelic losses are associated with a significant proportion (25%) of MSI- colorectal tumours, but are not involved in the metastatic process. PTEN and BMPR1A, two genes located in the common deleted region, were screened for mutations in samples with loss of heterozygosity. The absence or low frequency of mutations indicates that the inactivation of these genes by deletion of one allele and mutation of the other one plays only a minor role in MSI- tumours.

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.

Evaluation of mutation screening by heteroduplex analysis in acute intermittent porphyria: comparison with denaturing gradient gel electrophoresis.

Acute intermittent porphyria is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen deaminase (PBGD). Many different strategies have been developed to screen for mutations. However the high prevalence (0.6 per thousand) of PBGD gene defect, the large allelic heterogeneity of mutations (n = 130), and the limitations of the PBGD enzymatic assay for asymptomatic patients' detection, require for diagnosis an efficient and easy to handle strategy for locating mutations within the PBGD gene. In a recent study the sensitivity of the denaturing gradient gel electrophoresis (DGGE) technique was 100%. However DGGE requires the preparation of gradient gels and the use of primers with long GC-clamps; thus alternative methods should be preferable in the clinical laboratory. We have compared the detection rate of DGGE with heteroduplex analysis (HA) using 16 characterized PBGD gene mutations. Six different HA conditions were used to determine the efficiency of the method, including: (1) MDE (mutation detection enhancement) gel concentration; (2) addition of urea and sodium dodecyl sulfate (SDS); (3) radioactive labelling. The sensitivity of each HA condition varied from 31 to 81% vs. 100% in DGGE analysis. HA using 1 x MDE with 15% urea with or without 0.55% SDS was the most sensitive condition. This first comparative study of DGGE and HA mutation screening methods suggests that DGGE is a more sensitive screening assay than optimized HA. However, because of its simplicity HA should be considered as an efficient alternative mutation screening method.

New mutations of the hydroxymethylbilane synthase gene in German patients with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a low-penetrant, autosomal dominant disorder caused by decreased activity of hydroxymethylbilane synthase (HMBS; MIM 176 000), the third enzyme in the heme biosynthetic pathway. We report the first molecular analysis of HMBS gene mutations in classical AIP patients of German origin. The HMBS gene of 5 German AIP patients was analysed by DGGE-screening and direct sequencing of amplified genomic DNA. Five different mutations including four novel mutations were found. Three of them are single base substitutions that affected exon 3 (R16C), exon 10 (V202L), and intron 13 (T to A, IVS13+2) The two remaining mutations are frameshifts which produce a stop codon (del GA in exon 6 and insA in exon 14). These mutations are likely to be responsible for the decrease in HMBS activity found in both erythrocytes and non-erythroid cell lines (lymphocytes). Our results demonstrate the allelic heterogeneity of HMBS mutations in AIP patients of German origin.

Exon 1 donor splice site mutations in the porphobilinogen deaminase gene in the non-erythroid variant form of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial defect of the heme biosynthesis enzyme, porphobilinogen deaminase (PBGD). PBGD is encoded by two distinct mRNA species expressed in a tissue-specific manner from a single gene. One transcript is expressed in erythroid tissues, while the housekeeping transcript is expressed in all tissues. In classical AIP (95% of cases) the housekeeping and the erythroid-specific enzymes both have half-normal activity in erythroid and non-erythroid tissues, whereas in the variant non-erythroid form of the disease the enzymatic defect is present only in non-erythroid cells. A large allelic heterogeneity of mutations (n>135) has been demonstrated in classical AIP, but to date only three different mutations have been characterized in the non-erythroid variant form of AIP. We describe the molecular abnormalities responsible for the non-erythroid variant form of AIP in two French and two German unrelated AIP patients with normal PBGD activity in the erythrocytes. Three different splicing defects located in the intron 1 donor splice site were identified: a 33+1 g-->a mutation, previously described in a Dutch family, was found in two patients; two novel mutations (33+2 t-->a, 33+5 c-->g) affected the two remaining patients. All the mutations resulted in the activation of a cryptic splice site 67 bp downstream in intron 1, leading to a frameshift and a premature stop codon in exon 4. Mutations in the exon 1 donor splice site are involved in eight of the nine non-erythroid variant AIP families reported in the literature. These data show that most mutations causing the non-erythroid variant AIP are exon 1 splice defects, in contrast with classical AIP, where missense mutations are chiefly involved. Moreover, the allelic heterogeneity of PBGD gene defects causing the non-erythroid variant AIP is demonstrated, with five different mutations identified. These mutations could be easily detected by a single denaturing gradient gel electrophoresis which also allows the presymptomatic detection of gene carriers in the affected families.

Acute intermittent porphyria: prevalence of mutations in the porphobilinogen deaminase gene in blood donors in France.

OBJECTIVES: Acute intermittent porphyria (AIP) is an autosomal dominant disorder resulting from a 50% deficiency in porphobilinogen deaminase (PBG deaminase). The true prevalence in the general population of mutations in the PBG deaminase gene capable of causing AIP is unknown. However, it is important to identify asymptomatic carriers of AIP mutations because all are at risk to have an acute attack. DESIGN: We measured erythrocyte PBG deaminase from 3350 healthy blood donors. When a clear cut deficiency (< mean minus 2.5 SD) was found, the PBG deaminase gene was analysed by molecular biology technics. SUBJECTS: Four subjects with PBG deaminase deficiency were identified. Two had mutations in the PBG deaminase gene which are known to cause AIP. CONCLUSION: We conclude that, in France, the mutations of the PBG deaminase gene show a high prevalence in the healthy population. If only these two confirmed latent cases are used for the calculation, in France the minimal prevalence of the AIP gene is 1:1675.

Molecular epidemiology and diagnosis of PBG deaminase gene defects in acute intermittent porphyria.

Acute intermittent porphyria (AIP) is the major autosomal dominant form of acute hepatic porphyrias. The disease is due to mutations in the gene encoding for porphobilinogen (PBG) deaminase and is characterized by life-threatening neurovisceral attacks, often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. This report describes a prospective study on the molecular epidemiology of PBG deaminase gene defects in AIP. It uses a sensitive, reliable, and easy-to-handle method for routine AIP molecular diagnosis and family study based on an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing. Fifteen genomic DNA fragments, including all the coding sequence and covering 3.35 kb of the PBG deaminase gene, were investigated in 405 subjects from 121 unrelated French Caucasian AIP families who had not been screened previously at the DNA level. PBG deaminase gene mutations were identified in 109 families, but only 78 were of different type, and each of them had a prevalence rate < 5%. Among these mutations, 33 had not been published previously. Sixty percent of these 78 mutations were located in only three exons (exons 10, 12, and 14), 44% were missense, 18% were splice defect, 19% were frameshift, and 16% were nonsense. In addition, two de novo mutational events were characterized. The evaluation of the efficiency of the standard PBG deaminase enzymatic screening method for gene-carrier detection indicated 95% of concordancy with the molecular-based diagnosis.

Acute intermittent porphyria: rapid molecular diagnosis.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial porphobilinogen (PBG) deaminase deficiency. An exon-by-exon denaturing gradient gel electrophoresis (DGGE) analysis followed by direct sequencing of the DNA fragments was performed to investigate molecular defect in 8 unrelated patients living in south of France: one Algerian, two Moroccan and five French patients. We have optimized the DGGE method in order to study at the same time the fifteen exons of the PBG deaminase gene in only one electrophoresis run. Six different mutations were detected by abnormal mobility patterns. After characterization, a C insertion (716 ins C), 2 deletions (589 del 17 bp; 730 del CT), a non-sense mutation (R149X) and 2 missense mutations (A270G; R173W) were found. The R173W missense mutation was found in 3 unrelated patients, and 716 ins C, 589 del 17 bp and A270G were newly described. According to this small AIP samples, sensitivity of the DGGE screening method was 100%.

Protoporphyrinogen oxidase: complete genomic sequence and polymorphisms in the human gene.

Variegate porphyria (VP) is an autosomal dominant disorder of heme synthesis caused by a partial deficiency of protoporphyrinogen oxidase (PPOX). Human cDNA encoding PPOX has been recently sequenced and the gene has been cloned, assigned to chromosome 1q23, and its exon/intron organization has been characterized. We report here the complete nucleotide sequence of the Human PPOX gene. Including 660 bp of its promotor region, the PPOX gene spans 5.5 kb. Introns vary in size from 84 bp to 507 bp. Two exonic and 3 intronic biallelic sequence variations have been characterized.

Detection of four novel mutations in the porphobilinogen deaminase gene in French Caucasian patients with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder characterized by alterations of the gene encoding porphobilinogen deaminase (PBGD: EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway. The molecular heterogeneity of the mutations causing AlP has been demonstrated with a reported predominance of single base substitutions resulting in amino acid changes. The molecular basis of AIP in four French patients was investigated using denaturing gradient gel electrophoresis followed by direct sequencing. We describe four different novel mutations that affected exon 12 (a frameshift and an exon skipping), exon 4 (a stop codon) and exon 15 (a frameshift inducing a stop codon). This study further documents the molecular heterogeneity of mutations in the PBGD gene in the French Caucasian population and reports types of mutations relatively uncommon in AIP.

Mutations in the protoporphyrinogen oxidase gene in patients with variegate porphyria.

Variegate porphyria (VP) is an acute hepatic porphyria with autosomal dominant inheritance due to a partial deficiency of protoporphyrinogen oxidase (PPOX) activity. The molecular defect responsible for VP was investigated by sequencing PPOX gene coding sequence from four patients in three unrelated VP families of French Caucasian origin. In a first patient, a point insertion of a G at position 1022 of the cDNA, produced a frameshift resulting in a premature stop codon. In three other patients from two unrelated families we found a missense point mutation leading to glycine to arginine substitution (G232R) in exon 7. This Gly232 appears to be a strictly conserved residue through evolution. In one VP family, we observed the cosegregation of the G232R missense mutation and the deficient PPOX activity. The mutations reported here are the first to be described in patients with VP and support the conclusion that PPOX gene defects are disease causing mutations in human variegate porphyria.