Recurrent attacks of acute hepatic porphyria: major role of the chronic inflammatory response in the liver.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem metabolism characterized by life-threatening acute neurovisceral attacks due to the induction of hepatic δ-aminolevulinic acid synthase 1 (ALAS1) associated with hydroxymethylbilane synthase (HMBS) deficiency. So far, the treatment of choice is hemin which represses ALAS1. The main issue in the medical care of AIP patients is the occurrence of debilitating recurrent attacks. OBJECTIVE: The aim of this study was to determine whether chronic hemin administration contributes to the recurrence of acute attacks. METHODS: A follow-up study was conducted between 1974 and 2015 and included 602 French AIP patients, of whom 46 had recurrent AIP. Moreover, we studied the hepatic transcriptome, serum proteome, liver macrophage polarization and oxidative and inflammatory profiles of Hmbs-/- mice chronically treated by hemin and extended the investigations to five explanted livers from recurrent AIP patients. RESULTS: The introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. Moreover, we showed that both in animal model and in human liver, frequent hemin infusions generate a chronic inflammatory hepatic disease which induces HO1 remotely to hemin treatment and maintains a high ALAS1 level responsible for recurrence. CONCLUSION: Altogether, this study has important impacts on AIP care underlying that hemin needs to be restricted to severe neurovisceral crisis and suggests that alternative treatment targeting the liver such as ALAS1 and HO1 inhibitors, and anti-inflammatory therapies should be considered in patients with recurrent AIP.

Influence of meteorological data on sun tolerance in patients with erythropoietic protoporphyria in France.

BACKGROUND: Erythropoietic protoporphyria (EPP) is a rare metabolic disorder, characterized by photosensitivity, caused by errors of the haem biosynthetic pathway. Avoidance of sun exposure is recommended; however, some patients suggested a paradoxical improvement of symptoms when they move to sunny areas. OBJECTIVES: In a national French study, we sought to investigate the influence of sun exposure on EPP symptoms. MATERIALS AND METHODS: We used a national transversal observational study by questionnaire. Patients were selected from the national record of the Centre Français des Porphyries (French Porphyrias referral centre). Sun exposure level by geographic area was assessed using climate data provided by the French national meteorological service (Météo France). RESULTS: Eighty-nine patients were included. We notably observed that 40% of patients declared an improvement in their tolerance of sun exposure after repeated sun exposures. In the more sunny areas, the intensity of the pain was lower (r = -0·26) and the duration of the sun exposure responsible for flares was longer (r = 0·39) than in the areas that were less sunny (P < 0·05). CONCLUSIONS: This study proposes a benefit of natural progressive sun exposure for patients with EPP.

[Porphyrias and haem related disorders]. / Les porphyries héréditaires: anomalies du métabolisme de l'hème.

The hereditary porphyrias comprise a group of eight metabolic disorders of the haem biosynthesis pathway characterised by acute neurovisceral symptoms, skin lesions or both. Each porphyria is caused by abnormal function of a separate enzymatic step resulting in a specific accumulation of haem precursors. Seven porphyrias are the consequence of a partial enzyme deficiency while a gain of function mechanism has been recently characterised in a novel porphyria. Acute porphyrias present with severe abdominal pain, nausea, constipation, confusion and seizure, which may be life threatening. Cutaneous porphyrias can be present with either acute painful photosensitivity or skin fragility and blisters. Rare recessive porphyrias usually manifest in early childhood with either severe chronic neurological symptoms or chronic haemolysis and severe cutaneous photosensitivity. Porphyrias are still underdiagnosed, but once they are suspected, and depending on the clinical presentation, a specific and simple front line test allows the diagnosis in all symptomatic patients. Diagnosis is essential to institute as soon as possible a specific treatment. Screening families to identify presymptomatic carriers is crucial to prevent chronic complications and overt disease by counselling on avoiding potential precipitants.

From tissue iron retention to low systemic haemoglobin levels, new pathophysiological biomarkers of human abdominal aortic aneurysm.

Iron deposits are observed in tissue of abdominal aortic aneurysm (AAA) patients, although the underlying mechanisms are not completely elucidated. Therefore we explored circulating markers of iron metabolism in AAA patients, and tested if they could serve as biomarkers of AAA. Increased red blood cell (RBC)-borne iron retention and transferrin, transferrin receptor and ferritin expression was observed in AAA tissue compared to control aorta (immunohistochemistry and western blot). In contrast, decreased circulating iron, transferrin, mean corpuscular haemoglobin concentration (MCHC) and haemoglobin concentration, along with circulating RBC count, were observed in AAA patients (aortic diameter >3 cm, n=114) compared to controls (aortic diameter <3 cm, n=88) (ELISA), whereas hepcidin concentrations were increased in AAA subjects (MS/MS assay). Moreover, iron, transferrin and haemoglobin levels were negatively, and hepcidin positively, correlated with aortic diameter in AAA patients. The association of low haemoglobin with AAA presence or aortic diameter was independent of specific risk factors. Moreover, MCHC negatively correlated with thrombus area in another cohort of AAA patients (aortic diameter 3-5 cm, n=357). We found that anaemia was significantly more prevalent in AAA patients (aortic diameter >5 cm, n=8,912) compared to those in patients with atherosclerotic aorto-iliac occlusive disease (n=17,737) [adjusted odds ratio=1.77 (95% confidence interval: 1.61;1.93)]. Finally, the mortality risk among AAA patients with anaemia was increased by almost 30% [adjusted hazard ratio: 1.29 (95% confidence interval: 1.16;1.44)] as compared to AAA subjects without anaemia. In conclusion, local iron retention and altered iron recycling associated to high hepcidin and low transferrin systemic concentrations could lead to reduced circulating haemoglobin levels in AAA patients. Low haemoglobin levels are independently associated to AAA presence and clinical outcome.

What do French patients and geneticists think about prenatal and preimplantation diagnoses in Marfan syndrome?

OBJECTIVES: Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with manifestations mainly involving the skeletal, ocular, and cardiovascular systems. The phenotypic variability observed in MFS makes genetic counselling difficult. Prenatal diagnosis (PND) and preimplantation genetic diagnosis are technically feasible when a causal mutation is identified, but both raise many ethical questions in this condition. Little is known about opinions and practices in such reproductive issues in MFS. The goal of this study was to report on patients' points of view and geneticists' standard practices. METHODS: Two different questionnaires were produced. RESULTS: Fifty geneticists filled in the questionnaire. Twenty-two per cent thought that PND was acceptable, 72% debatable and 6% not acceptable. Preimplantation genetic diagnosis was more often reported acceptable (34% of answers). Results varied according to the physician's experience with the disease. Fifty-four answers were collected for patients' questionnaires. Most of them (74%) were favourable to the development of prenatal testing, and believed that the choice should be given to parents. However, only a minority would opt for prenatal diagnosis for themselves. CONCLUSION: This study showed that the majority of patients were in favour of PND and that opinions among practitioners varied widely, but that overall, practitioners favoured a systematic multidisciplinary evaluation of the couple's request.

ACVRL1 germinal mosaic with two mutant alleles in hereditary hemorrhagic telangiectasia associated with pulmonary arterial hypertension.

Germline mutations in genes encoding members of the transforming growth factor-ß (TGF-ß)/bone morphogenetic protein (BMP) superfamily are causal for two hereditary vascular disorders, hereditary hemorrhagic telangiectasia (HHT) and heritable pulmonary arterial hypertension (PAH). When the two diseases coexist, activin A receptor type II-like kinase-1 (ACVRL1) gene mutations are usually identified. We report a remarkable ACVRL1 germinal and somatic mosaicism characterized by the presence of two distinct mutant alleles and a non-mutant ACVRL1 allele in a woman diagnosed with PAH at the age 40. She also met the Curaçao diagnostic criteria for HHT based on additional findings of telangiectases, epistaxis and arteriovenous malformations. Mutation analysis of ACVRL1 identified two adjacent heterozygous deleterious mutations within exon 10: c.1388del (p.Gly463fsX2) and c.1390del (p.Leu464X) in a region enriched by mutation-associated DNA motifs. The mother transmitted the c.1388del to one child and the c.1390del to two children confirming germinal mosaicism. Allele-specific polymerase chain reaction analysis showed that c.1388del is the predominant mutation in lymphocytes of the index case. Haplotype analysis revealed that both mutant alleles have a common chromosomal origin which is distinct from that of the mother's non-mutant ACVRL1 allele. These distinct mutant alleles in tissues and germline could have arisen by DNA structure-mediated events occurring in the early stages of the mother's embryogenesis, prior to the segregation of her germline, which ultimately led to the independent transmission of each allele. These highlight the complexity of genomic events occurring during early embryogenesis and the consequences of mutational mosaicism upon pathogenic variability.

[Inheritance in erythropoietic protoporphyria]. / La protoporphyrie érythropoïétique: une maladie, deux gènes et trois mécanismes moléculaires.

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis that results from an accumulation of protoporphyrin IX in erythroid cells, plasma, skin and liver. EPP leads to acute photosensitivity and, in about 2% of patients, liver disease. EPP is a complex syndrome in which two genes are independently involved: FECH and ALAS2. More than 96% of unrelated EPP patients have ferrochelatase (FECH) deficiency (MIM 177000). Four percent of them present with autosomal recessive inheritance with two mutated FECH alleles. In dominant cases (95%) the inheritance of a common hypomorphic IVS3-48C FECH allele trans to a deleterious FECH mutation reduces FECH activity below a critical threshold. The frequency of the IVS3-48C allele differs widely from the Japanese (45%), to Black West Africans (<1%) populations. These differences in the frequency of this single common SNP account for the prevalence of overt EPP in different countries and for the absence of EPP in Black Africans. The phylogenic origin of the IVS3-48C haplotypes strongly suggests that the IVS3-48C allele arose from a single recent mutational event that occurred 60 Kyears ago. Acquired somatic mutation of FECH secondary to myeloid disease may also exceptionally cause EPP (<1%). Finally, about 4% of unrelated EPP patients have X-linked dominant protoporphyria (XLDPP) (MIM 300752) caused by gain-of-function mutations in the ALAS2 gene leading to an increased erythroid heme biosynthesis and subsequently an accumulation of protoporphyrin without any FECH deficiency.

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.

Excessive erythrocyte PPIX influences the hematologic status and iron metabolism in patients with dominant erythropoietic protoporphyria.

Partial deficiency of the last enzyme of the heme biosynthetic pathway (namely ferrochelatase, FECH) in humans is responsible for erythropoietic protoporphyria (EPP). This disorder is characterised by painful photosensitivity, due to excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial hypotheses have been proposed to explain the hematologic and iron status of EPP patients. In the present work, we explored these parameters in 55 patients with dominant EPP recruited at the French Center of Porphyrias (Colombes, France) and confirmed by molecular analysis. Our data show that erythrocyte accumulation of PPIX in EPP patients influences hematologic and iron status. Patients studied had a mild anemia and thrombocytopenia, as shown by the downward shift of hematologic parameters, which positively correlated with the amount of erythrocyte PPIX. Interestingly, erythropoiesis did not seem to be limited by iron supply in patients, since serum iron and soluble transferring (Tf) receptor (sTfR) were normal. However, iron and Tf saturation negatively correlated with erythrocyte PPIX. Moreover, and as previously described in a mouse model of EPP, we noted a positive correlation between erythrocyte PPIX and Tf levels. Altogether, these results suggest a positive effect of PPIX on the synthesis on Tf, which could facilitate the mobilization of tissue iron stores to meet erythropoiesis requirement. Based on these observations and previous results in EPP mouse model, we propose that the PPIX-liver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen and the bone marrow.

Impulsiveness as the intermediate link between the dopamine receptor D2 gene and alcohol dependence.

OBJECTIVES: Reinforcement and reward processes have been proposed as being an intermediate link between the risk for alcohol dependence and the gene coding for the dopamine receptor D2 (DRD2). This hypothesis remains open to speculation, and personality traits such as impulsiveness, a core dimension in addictive disorders, should also be taken into account. For instance, recent evidence in rats showed that DRD2 antagonists might increase impulsivity in decreasing the value of delayed rewards. METHODS: Considering the pro-impulsiveness role of ethanol observed in clinical practice and epidemiological studies, we analysed the Barratt impulsiveness scores in a sample of 92 alcohol-dependent French patients (57 men and 35 women), according to the TaqI A polymorphism of the DRD2 gene. RESULTS: A2/A2 and A1/A2 genotypes were significantly associated with a higher global impulsiveness than A1/A1 genotype (P=0.02 and P=0.03, respectively). CONCLUSIONS: We propose that reward-related impulsiveness may constitute a risk factor for alcohol dependence, and that this core temperament could be partly mediated by the DRD2 gene.

A genotype-phenotype correlation between null-allele mutations in the ferrochelatase gene and liver complication in patients with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP), an inborn error of heme metabolism, causes in the majority of the patients only a symptom of photosensitivity. However, around 2% of the EPP sufferers develop liver complication in the form of liver cirrhosis and progressive liver failure. Mutations in the human ferrochelatase (FECH) gene causing EPP are highly heterogeneous and mostly family-specific. Actually, 62 FECH mutations have been published, 48 of them are "null allele" mutations inducing the formation of a truncated protein. The remaining 14 are missense mutations. In contrast to the null allele mutations, the latter lead to substitution of a single amino acid residue in the protein molecule and generate an enzyme that, although functionally impaired, is in its full length. In order to study the association between "null allele" mutation and liver complication, we combined our data with those in the literature. A total of 112 EPP patients were counted among 93 EPP families with a known FECH mutation. All 18 EPP patients who had severe liver complication carried a "null allele" mutation. In contrast, none of the 20 patients who carried a missense mutation had developed liver complication till the time of study (Fisher's exact test, p<0.05). High protoporphyrin blood concentration are considered to be a sign of an increased risk of liver disease. No correlation of protoporphyrin blood level with the type of mutation, was found, if patients with overt liver disease were excluded from the sample. Furthermore, no significant association of the liver complication with the location of the mutation within the FECH gene was found (Fisher exact test p = 0.46). These available data indicate a significant genotype-phenotype correlation between "null allele" mutation and protoporphyrin related liver disease in EPP. Although the risk for a EPP patient with a missense mutation to develop liver disease cannot be totally eliminated based on these data, it is comparably low.

Association and excess of transmission of a DRD2 haplotype in a sample of French schizophrenic patients.

The gene which codes for dopamine receptor D2 (DRD2) is considered as one of the most relevant candidate genes in schizophrenia. Previous genetic studies focusing on this gene gave conflicting results, potentially because of the differences in methodology (linkage versus association studies), and the different loci analyzed (the DRD2 gene having many polymorphisms). We used a progressive strategy with three different approaches (case/control, haplotype relative risk and transmission disequilibrium test) and investigated two genetic polymorphisms (TaqI B1/B2 and TaqI A1/A2, spanning the coding region of the DRD2 gene) in 50 patients with DSM-IV diagnoses of schizophrenia, in their 100 parents and in 50 healthy, matched controls. Firstly, we found a significant excess of the two alleles (B2 and A2) in the schizophrenic group compared to unaffected controls. Secondly, we found an excess of transmission from the parents to their affected children, using the haplotype relative risk design applied to the B2A2 haplotype. Finally, the transmission disequilibrium test showed evidence for linkage between B2A2 haplotype and schizophrenia. The significant excess of the B2A2 haplotype in schizophrenic patients is specifically observed in a subsample of patients with a disease onset occurring after 20 years of age. As the haplotype contains nearly the entire DRD2 gene, we found convergent evidence in our sample for a significant role of the DRD2 gene in the risk for schizophrenia. This haplotype may be more specifically involved in the disorder's onset at a later age in some patients, or, alternatively, may be implicated as a modifying factor acting on age of onset.

The genetics of addiction: alcohol-dependence and D3 dopamine receptor gene.

Alcohol-dependence is a complex phenotype, with behavioral, psychological, pharmacological, medical and social dimensions. Aggregation studies, adoption and twin researches have demonstrated that the vulnerability to alcohol-dependence is at least in part linked to genetic factors, the genetic vulnerability to alcoholism being mainly not substance-specific. There are numerous candidate genes, but the D3 dopamine receptor is specifically located in the limbic area, and in particular in the nucleus accumbens, which are involved in reward and reinforcement behavior. Furthermore, a previous collaborative study showed that homozygosity for the Ball DRD3 locus was more frequently observed in opiate dependent patients with high sensation seeking scores. In this study, we analyzed the distribution of Ball DRD3 polymorphism in a new sample of 131 French male alcoholic-patients (DSM III-R criteria) and 68 healthy controls matched for sex and origins. Although we replicated the higher sensation seeking score in alcohol-dependent patients with comorbid dependence, we found no significant difference in the DRD3 gene polymorphism between controls and alcoholic patients, regardless of sensation seeking score, addictive or psychiatric comorbidity, alcoholism typology, and clinical specificities of alcoholism. There is good evidence that gene coding for the dopamine receptor D3 does not play a major role in the genetic vulnerability to alcoholism.

New insights into the pathogenesis of erythropoietic protoporphyria and their impact on patient care.

UNLABELLED: Erythropoietic protoporphyria (EPP, MIM 177000) is an inherited disorder caused by a partial deficiency of ferrochelatase (FECH) which catalyses the chelation of iron into protoporphyrin to form haem. The majority of EPP patients experience solely a painful photosensitivity whereas a small number of them develop liver complications due to the accumulation of excessive amount of protoporphyrin in the liver. EPP is considered to be an autosomal dominant disorder, however, with a low clinical penetrance. To date, a total of 65 different mutations have been identified in the FECH gene of EPP patients. Among the 89 EPP patients who carry a "null allele" mutation which results in the formation of a truncated protein, 18 of them developed EPP-related liver complications. None of the 16 missense mutations identified among 19 patients on the other hand, have been associated with liver disease (P = 0.038). The allelic constellation of an overt patient consists of a mutated FECH allele and a "low expressed" normal allele and that of an asymptomatic carrier, a combination of a mutated and a normally expressed FECH allele. The identification of the "low expressed" allele is facilitated by haplotype analysis using two single nucleotide polymorphisms, -251 A/G in the promoter region and IVS1-23C/T. At the current time when only partially effective therapies are available, the disclosures of both "null allele" and the "low expression" mechanisms will improve patient management. CONCLUSION: While covering the important clinical aspect of erythropoietic protoporphyria, this article emphasises the latest achievements in the molecular genetics of the disorder.

Mutations in the iron-sulfur cluster ligands of the human ferrochelatase lead to erythropoietic protoporphyria.

Ferrochelatase (FECH; EC 4.99.1.1) catalyzes the terminal step of the heme biosynthetic pathway. Defects in the human FECH gene may lead to erythropoietic protoporphyria (EPP), a rare inherited disorder characterized by diminished FECH activity with protoporphyrin overproduction and subsequent skin photosensitivity and in rare cases liver failure. Inheritance of EPP appeared to be autosomal dominant with possible modulation by low expression of the wild-type FECH allele. Animal FECHs have been demonstrated to be [2Fe-2S] cluster-containing proteins. Although enzymatic activity and stability of the protein appear to be dependent on the presence of the [2Fe-2S] cluster, the physiologic role of the iron-sulfur center remains to be unequivocally established. Three of the 4 [2Fe-2S] cluster-coordinating cysteines (ie, C403, C406, and C411 in the human enzyme) are located within the C-terminal domain. In this study 5 new mutations are identified in patients with EPP. Three of the point mutations, in 3 patients, resulted in FECH variants with 2 of the [2Fe-2S] cluster cysteines substituted with tyrosine, serine, and glycine (ie, C406Y, C406S, and C411G) and with undetectable enzymatic activity. Further, one of the patients exhibited a triple point mutation (T(1224)-->A, C(1225)-->T, and T(1231)-->G) leading to the N408K/P409S/C411G variant. This finding is entirely novel and has not been reported in EPP. The mutations of the codons for 2 of the [2Fe-2S] cluster ligands in patients with EPP supports the importance of the iron-sulfur center for the proper functioning of mammalian FECH and, in at least humans, its absence has a direct clinical impact. (Blood. 2000;96:1545-1549)

Reappraisal of the association between the DRD2 gene, alcoholism and addiction.

We analysed the impact of the TaqI A1 allele of the D2 dopamine receptor gene on the risk for alcoholism, trying to depict three explanations frequently proposed to explain discrepancies in association and linkage studies: that the A1 allele may act as a marker rather than as a vulnerability factor, that stratification biases and unevaluated controls may explain positive results, and that the A1 allele is modifying the phenotype rather than increasing the risk for alcoholism. We thus tested another (dinucleotide STRP) marker within the DRD2 gene, selected a new homogenous sample of 113 alcoholic patients and 49 unaffected controls strictly matched for ethnic origins, and systematically assessed both samples with a semi-structured interview to detect (in both samples) alcohol dependence, but also such related traits as specificities of complications. The frequency of the A1 allele was not significantly different between alcoholics and controls but when comparing different subgroups of alcoholics, the A1 allele was significantly more frequent in alcoholic patients with somatic complications (OR = 3.00, CI[1.37-6.62]), social and professional complications (OR = 2. 72, CI[1.25-5.90]), or with co-morbid dependence (OR = 2.88, 95% IC [1.16-7.15]). The association for co-morbid dependence and somatic complications was also positive when taking into consideration both STRP and TaqIA polymorphisms. The A1 allele does not increase the risk for alcoholism per se in our sample, but may be involved in a related trait which is partially dependent on the diagnosis of alcoholism, through a disequilibrium with another close mutation.

Inheritance in erythropoietic protoporphyria: a common wild-type ferrochelatase allelic variant with low expression accounts for clinical manifestation.

Erythropoietic protoporphyria (EPP) is a rare autosomal dominant disorder of heme biosynthesis characterized by partial decrease in ferrochelatase (FECH; EC 4.99.1.1) activity with protoporphyrin overproduction and consequent painful skin photosensitivity and rarely liver disease. EPP is normally inherited in an autosomal dominant pattern with low clinical penetrance; the many different mutations that have been identified are restricted to one FECH allele, with the other one being free of any mutations. However, clinical manifestations of dominant EPP cannot be simply a matter of FECH haploinsufficiency, because patients have enzyme levels that are lower than the expected 50%. From RNA analysis in one family with dominant EPP, we recently suggested that clinical expression required coinheritance of a normal FECH allele with low expression and a mutant FECH allele. We now show that (1) coinheritance of a FECH gene defect and a wild-type low-expressed allele is generally involved in the clinical expression of EPP; (2) the low-expressed allelic variant was strongly associated with a partial 5' haplotype [-251G IVS1-23T IVS2microsatA9] that may be ancestral and was present in an estimated 10% of a control group of Caucasian origin; and (3) haplotyping allows the absolute risk of developing the disease to be predicted for those inheriting FECH EPP mutations. EPP may thus be considered as an inherited disorder that does not strictly follow recessive or dominant rules. It may represent a model for phenotype modulation by mild variation in expression of the wild-type allele in autosomal dominant diseases.

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.

Systematic analysis of coproporphyrinogen oxidase gene defects in hereditary coproporphyria and mutation update.

Hereditary coproporphyria (HC) is an acute hepatic porphyria with autosomal dominant inheritance caused by deficient activity of coproporphyrinogen III oxidase (CPO). Clinical manifestations of the disease are characterized by acute attacks of neurological dysfunction often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases. Skin photosensitivity may also be present. The seven exons, the exon/intron boundaries and part of 3' noncoding sequence of the CPO gene were systematically analyzed by an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing in seven unrelated heterozygous HC patients from France, Holland, and Czech Republic. Seven novel mutations and two new polymorphisms were detected. Among these mutations: two are missense (G197W, W427R), two are nonsense (Q306X, Q385X), two are small deletions (662de14bp; 1168del3bp removing a glycine at position 390), and one is a splicing mutation (IVS1-15c-->g) which creates a new acceptor splice site. The pathological significance of the point mutations G197W, W427R, and the in-frame deletion 390delGly were assessed by their respective expression in a prokaryotic system using site-directed mutagenesis. These mutations resulted in the absence or a dramatic decrease of CPO activity. The two polymorphisms were localized in noncoding part of the gene: 1) a C/G polymorphism in the promotor region, 142 bp upstream from the transcriptional initiation site (-142C/G), and 2) a 6 bp deletion polymorphism in the 3' noncoding part of the CPO gene, 574 bp downstream of the last base of the normal termination codon (+574 delATTCTT). Five intragenic dimorphisms are now well characterized and the high degree of allelic heterogeneity in HC is demonstrated with seven new different mutations making a total of nineteen CPO gene defects reported so far.