May 2006 update in porphobilinogen deaminase gene polymorphisms and mutations causing acute intermittent porphyria: comparison with the situation in Slavic population

Acute intermittent porphyria (AIP) is an autosomal dominant disorder of heme biosynthesis caused by molecular defects in the porphobilinogen deaminase (PBGD) gene. This paper reviews published mutations, their types, and polymorphisms within the PBGD gene. To date, 301 different mutations and 21 polymorphisms have been identified in the PBGD gene in AIP patients and individuals from various countries and ethnic groups. During the search for mutations identified among Slavic AIP patients we found 65 such mutations and concluded that there is not a distinct predominance of certain mutations in Slavs.     

Porphobilinogen deaminase gene in African and Afro-Caribbean ethnic groups: mutations causing acute intermittent porphyria and specific intragenic polymorphisms

Acute intermittent porphyria (AIP), the most common acute hepatic porphyria, is a low-penetrant autosomal dominant disorder caused by mutations in the porphobilinogen deaminase (PBGD) or hydroxymethylbilane synthase (HMBS) gene. Although AIP has been identified in all the main ethnic groups, little is known about PBGD gene defects in Africans, Afro-Caribbean and Afro-Americans. We have carried out PBGD gene screening among seven unrelated AIP families and 98 controls belonging to the Afro-Caribbean (French West Indies) and the sub-Saharan African (Morocco, Algeria, Cameroon, Mali, and Burkina Faso) populations. Using denaturing-gradient gel electrophoresis (DGGE) and direct sequencing we characterized six different mutations, including four novel, from the seven AIP families: three splicing defects (IVS 5+2 Ins G; IVS 7+1 G to A in two families; IVS 10-1 G to T); a small deletion (1004 Del G); and two missense mutations (R116 W; A270G). The allele frequencies of the 14 polymorphic sites, previously known in the normal Caucasian population, were similar in Africans and Afro-Caribbean control populations. Interestingly, two common new intragenic polymorphic sites, close to intron/junction boundaries, were identified only in blacks: 1) in intron 2, a single base-pair G deletion at position 3167 (G:0.88; delG:0.12); 2) in intron 10, a A/G dimorphism at position 7052 (A:0.56; G:0.44). These two single nucleotide polymorphisms (SNPs) were never encountered in 750 unrelated Caucasian subjects. The allele frequency distributions of populations within black ethnic groups (Africans and Afro-Caribbean) are similar. This study highlights differences both in PBGD gene mutations causing AIP and in SNPs between white and black peoples; the allele frequencies provided contribute to a better knowledge of the variability of these markers among the major population groups, especially in sub-Saharan West African and Afro-Caribbean populations.     

Molecular genetic study of acute intermittent porphyria in Russia: Mutation analysis and functional polymorphism search in porphobilinogen deaminase gene

Acute intermittent porphyria (AIP) is an autosomal dominant hereditary disease, caused by partial deficiency of porphobilinogen deaminase (PBGD), one of the key enzymes of heme biosynthesis. This study describes molecular genetics of AIP in Russia. Mutation analysis of PBGD gene in 70 unrelated patients revealed 47 various genetic defects, 28 of which had not been described previously. Mutations 53delT and Arg173Trp (recorded 8 times, in total 23%) proved to be the most common in Russia. Microdeletion 53delT has monophyletic origin and was found only in Russia. Molecular genetic examination of 132 relatives of AIP patients from 40 families revealed 52 latent carriers of the disease. Low (about 10%) AIP penetrance indicates that a mutation in the PBGD gene is an important but not sufficient prerequisite for clinical manifestation of the disease. Modulation of penetrance in erythropoietic protoporphyria by coinheritance of a mutant allele and a functionally defective wild type allele of ferrochetalase gene has been shown previously. We hypothesized that similar mechanism works in AIP. Sequencing of the full length PBGD genes from unrelated AIP patients as well as SNP analysis, and the analysis of abnormal PBGD mRNA splicing showed that in case of AIP, this hypothesis is not true and some other factors are responsible for the penetrance of this disease.     

Correction of the biochemical defect in porphobilinogen deaminase deficient cells by non-viral gene delivery

Porphobilinogen deaminase (PBGD), the third enzyme in the biosynthesis of heme, is deficient in acute intermittent porphyria (AIP). AIP is a genetic disease characterized by neurovisceral and psychiatric disturbances. Despite a palliative treatment, it may still be lethal. An initial step towards gene therapy was recently taken by showing that PBGD could be expressed to correct the enzyme deficiency in AIP fibroblasts. The aim of the present study was to investigate whether the biochemical defect can be corrected by using non-viral gene delivery. The biochemical defect in human and mouse PBGD deficient fibroblasts was demonstrated by analyzing synthesis of the heme precursor, protoporphyrin (PP), after addition of 5-aminolevulinic acid (ALA). Human AIP fibroblasts synthesized 21% and mouse PBGD deficient fibroblasts only 11% of the PP amount synthesized in respective control cells. Gene delivery increased the PBGD activity 88–200 fold in human AIP fibroblasts and synthesis of PP was increased from 21–152% of normal after ALA incubation. Similar results were obtained in mouse PBGD deficient cells, although the PP levels were several-fold lower as compared to human cells. HPLC analysis confirmed that PP was the main porphyrin intermediate that was formed. Addition of porphobilinogen (PBG) resulted in 3–7 fold lower synthesis of PP as compared to ALA addition. These results show that non-viral gene delivery of plasmids encoding PBGD results in a high expression of functional PBGD shown by induced synthesis of PP in PBGD deficient cells after supplementation of ALA and PBG.     

Influence of age and gender on the clinical expression of acute intermittent porphyria based on molecular study of porphobilinogen deaminase gene among Swiss patients

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder in the heme biosynthetic pathway caused by a partial deficiency of porphobilinogen (PBG) deaminase. Clinically, AIP is characterized as acute neurovisceral attacks that are often precipitated by exogenous factors such as drugs, hormones, and alcohol. An early detection of mutation carriers is essential for prevention of acute attacks by avoiding precipitating factors. This study was aimed at analyzing genetic defects causing AIP among Swiss families to further investigate aspects concerning the clinical expression of the disease. MATERIALS AND METHODS: The PBGD gene of index patients from 21 Swiss AIP families was systematically analyzed by denaturing gradient gel electrophoresis of polymerase chain reaction (PCR) amplified DNA fragments and direct sequencing. RESULTS: Five new mutations insA503, del L170, T190I, P241S, and R321H, as well as three known mutations (R26H, R173Q and W283X) were detected. Twelve of the 21 index patients (57%) carried the prevalent mutation W283X previously found among the Swiss AIP population. Family-specific mutations were then screened among relatives of the index patients. Among the 107 studied individuals, 58 carried a PBGD gene mutation--30 were overt AIP patients and 28 were asymptomatic carriers. The apparent rate of overt disease in the study cohort was 52%, which is significantly higher than the previously reported penetrance of 10-20%. To further examine the clinical expression of AIP, the cumulative life-time risk was calculated among 58 mutation-positive individuals after stratifying for age. The result shows a linear increase of the percentage of the symptomatic patients with age, reaching up to 75% among carriers aged over 60. Moreover, statistical analysis of the gender distribution among patients and asymptomatic carriers indicated that the disease was more frequently expressed among females than males (Fisher's exact test two sided, p= (0.001). CONCLUSIONS: This comprehensive search for genetic defects in the PBGD gene confirmed the existence of a prevalent mutation W283X among Swiss AIP patients, as well as a number of family-private mutations. Genetic analysis laid a groundwork for further studies such as the effects of gender and age on the clinical expression of AIP.     

Three new mutations in the porphobilinogen deaminase gene, detected in acute intermittent porphyria patients from Russia]

Porphobilinogen deaminase (PBGD) is a key enzyme of the heme biosynthetic pathway. Defects in the PBGD gene lead to an autosomal dominant disease, acute intermittent porphyria (AIP). Almost all AIP patients with rare exceptions are heterozygous for the defective gene. To date, at least 160 different mutations causing AIP are identified. Extensive investigations along this line are conducted in many countries of the world. In Russia these studies had not been hitherto performed. Here we report the results of molecular genetic examination of four Russian patients with AIP diagnosed from clinical symptoms. By direct sequencing of the PBGD gene or the corresponding cDNA, we have detected four mutations, three of which were not previously encountered in the world population. These are TAAG deletion in intron 7 between positions +2 and (IVS7 2-5 delTAAG); T deletion in the initiation codon ATG of exon 3, and the G for C replacement at position -1 of intron 5 (IVS5 as -1 G:C), which disrupts splicing. In addition, in one female patient, a known deletion CT in codon 68 was revealed. In two patients, expression of PBGD gene alleles was significantly disproportional, so that normal mRNA prevailed in one case and mRNA of nonerythroid type in the other. Deletion in intron 7 was easily detectable due to the formation of a heteroduplex fragment with abnormal electrophoretic mobility directly in PCR. This simple heteroduplex analysis allowed us to exclude AIP carriage in son and daughter of a female patient with the genetic defect.     

Three Novel Mutations in Porphobilinogen Deaminase Gene Identified in Russian Patients with Acute Intermittent Porphyria

Porphobilinogen deaminase (PBGD) is a key enzyme of the heme biosynthetic pathway. Defects in the PBGD gene lead to an autosomal dominant disease, acute intermittent porphyria (AIP). Almost all AIP patients with rare exceptions are heterozygous for the defective gene. To date, at least 160 different mutations causing AIP are identified. Extensive investigations along this line are conducted in many countries of the world. In Russia these studies had not been hitherto performed. Here we report the results of molecular genetic examination of four Russian patients with AIP diagnosed from clinical symptoms. By direct sequencing of the PBGD gene or the corresponding cDNA, we have detected four mutations, three of which were not previously encountered in the world population. These are TAAG deletion in intron 7 between positions +2 and +5 (IVS7 2–5 delTAAG); T deletion in the initiation codon ATG of exon 3, and the G for C replacement at position –1 of intron 5 (IVS5 as –1 G–C), which disrupts splicing. In addition, in one female patient, a known deletion CT in codon 68 was revealed. In two patients, expression of PBGD gene alleles was significantly disproportional, so that normal mRNA prevailed in one case and mutant mRNA of nonerythroid type in the other. Deletion in intron 7 was easily detectable due to the formation of a heteroduplex fragment with abnormal electrophoretic mobility directly in PCR. This simple heteroduplex analysis allowed us to exclude AIP carriage in son and daughter of a female patient with the genetic defect.     

Ancestral founder of mutation W283X in the porphobilinogen deaminase gene among acute intermittent porphyria patients

Acute intermittent porphyria (AIP) is a low-penetrant autosomal dominant disorder caused by mutations in the porphobilinogen deaminase gene (PBGD). Nearly 60% of all Swiss AIP patients carry a nonsense mutation W283X (G(7916)-->A). In France, the prevalence of W283X is <5%. To determine whether W283X was a founder mutation or originated from multiple de novo events, we studied 25 apparently unrelated W283X families and index patients, 21 of Swiss and 4 of French origins. In the absence of sufficient genealogical data to verify the ancestral background of these W283X families/patients, we identified haplotypes of seven intragenic single nucleotide polymorphisms (SNPs) in the PBGD gene as well as eight microsatellites flanking the PBGD gene covering 9.88 cM in chromosome 11. Molecular cloning and sequencing experiments were required in order to completely resolve the intragenic haplotypes in this study cohort which mainly consisted of single index patients and families with limited members. Thirteen of the 25 W283X families/patients carry a SNP haplotype [C-A-A-A-G-C-W283X-G] and 12 (including four French families) carry a [T-G-G-G-G-C-W283X-G] haplotype. A less conserved microsatellite haplotype was identified among the 25 W283X alleles which allowed us to estimate the age of the mutation. Since W283X is not explained by a methylcytosine mutation, we favor the hypothesis of a single mutational event which took place on the [T-G-G-G-G-C-G] background at approximately 40 generations or 1000 years ago. Around 550 years ago, a recombination event occurred between intron 3 and 10 of the PBGD gene which resulted in the [C-A-A-A-G-C-W283X-G] haplotype only found in a restricted region.     

High prevalence of a point mutation in the porphobilinogen deaminase gene in Dutch patients with acute intermittent porphyria

Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by a deficiency of porphobilinogen deaminase (PBGD). Up to now 14 different mutations have been described. In an effort to investigate the molecular epidemiology of AIP we have undertaken a systematic study of different exons of the PBGD gene from a large number of unrelated patients. Here, exon 8 from 82 unrelated Dutch and French AIP patients was examined using single strand confirmation polymorphism analysis (SSCP) after polymerase chain reaction (PCR) amplification. A single base mutation, C to T, at position 346 of the sequence coding for PBGD was observed in 15 Dutch families but in only 1 French family. A simple PCR assay is described to facilitate the diagnosis of this common mutation at the DNA level.     

Identification of a prevalent nonsense mutation (W283X) and two novel mutations in the porphobilinogen deaminase gene of Swiss patients with acute intermittent porphyria

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by decreased activity of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthetic pathway. We report the first molecular analysis of PBGD gene mutations in AIP patients of Swiss origin. The PBGD gene of 18 Swiss AIP patients was analyzed by denaturing gradient gel electrophoresis screening of the genomic DNA and direct sequencing. Thirteen of the 18 patients (72%) carried a nonsense mutation G(849)-->A, W283X. In addition, 4 different mutations including 2 novel mutations (Q217L and Q292X), were identified in the 5 remaining AIP patients originating from both German- and Italian-speaking regions of Switzerland.     

De Novo mutation found in the porphobilinogen deaminase gene in Slovak acute intermittent porphyria patient: molecular biochemical study

The porphyrias are group of mostly inherited disorders in which a specific spectrum of accumulated and excreted porphyrins and heme precursors are associated with characteristic clinical features. There are eight enzymes involved in the heme synthesis and defects in seven of them cause porphyria. Four of them are described as acute hepatic porphyrias, which share possible precipitation of acute attacks with symptoms engaging the nervous system. Acute intermittent porphyria (AIP), caused by partial deficiency of the porphobilinogen deaminase (PBGD), is the most frequent among hepatic porphyrias. Clinical expression is highly variable and ~ 90 % of AIP heterozygotes remain asymptomatic throughout life. During systematic genetic analysis of the AIP patients diagnosed in the Czech and Slovak Republics, we found a special case of AIP. In a 15-year-old boy with abdominal and subsequent neurological symptomatology, we identified de novo mutation 966insA within the PBGD gene leading to a stop codon after 36 completely different amino acids compared to the wt-sequence. To establish the effects of this mutation on the protein structure, we expressed mutant constructs with described mutation in E. coli and analyzed their biochemical and enzymatic properties. Moreover, computer-assisted protein structure prediction was performed.     

Assignment of human porphobilinogen deaminase to 11q24.1----q24.2 by in situ hybridization and gene dosage studies

In situ hybridization and gene dosage-effect studies were conducted to determine the detailed chromosomal location of the gene encoding human porphobilinogen deaminase (PBGD). Red cell PBGD activity was normal in one patient with monosomy for 11q24.2----qter but was increased 1.5 times in another patient with trisomy for 11q22.2----qter. The cDNA probe for PBGD was found to be specifically hybridized to band 11q24. These results suggest that the gene for PBGD is localized within the region 11q24.1----q24.2.     

Two new mutations in the porphobilinogen deaminase gene and a screening method using PCR amplification of specific alleles

Acute intermittent porphyria (AIP) is attributable to defects in the porphobilinogen deaminase (PBGD) gene. Two new mutations have been found in the PBGD gene in Swedish families. The first is a G to A splice mutation in the last position of intron 9. A screening method using allele-specific amplification has been designed for the rapid detection of this mutation. The second mutation is a C to T substitution in exon 10, changing Arg²⁰¹ to Trp. This mutation can be detected by restriction enzyme cleavage.     

Detection of eleven mutations causing acute intermittent porphyria using denaturing gradient gel electrophoresis

Acute intermittent porphyria (AIP) is an autosomal dominant disease characterized by mutations of the gene coding for porphobilinogen deaminase (PBGD). Until now, sixteen different mutations have been described. In an effort to investigate further the molecular epidemiology of AIP, we have undertaken a systematic study of different exons of the PBGD gene from a large number of unrelated patients. Here, we have examined seven of the fifteen exons of the gene from 43 unrelated Dutch and French AIP patients using denaturing gradient gel electrophoresis after polymerase chain reaction amplification. Eleven new mutations were found, accounting for the enzymatic defect in about half of the patients. This study further documents the molecular heterogeneity of the mutations responsible for AIP and describes an efficient strategy to detect the mutations in patients with previously unknown abnormalities.     

Evidence for involvement of a second genetic locus on chromosome 11q in porphyrin metabolism

Chester porphyria is a distinct type of acute porphyria, which shows a biochemical overlap with acute intermittent and variegate porphyrias and has a dual enzyme deficiency of porphobilinogen deaminase (PBGD) and protoporphyrinogen oxidase. Linkage analysis in an extensive family with Chester porphyria was undertaken using multiple polymorphic markers. A maximum two point Lod score of 5.25 at 0.07 recombination (confidence interval 0.01 to 0.14) was observed with D11S351, which has been localised to 11q23.1. Multipoint linkage analysis confirmed the two point results and gave a maximum Lod score of 7.33 at a distance less than 1 cM proximal to D11S351. PBGD also maps to 11q but four recombinants could be identified from ten informative meioses in this family using a PBGD DNA polymorphism. Thus, a separate locus on 11q appears to be the basis of Chester porphyria.     

Identification of acute intermittent porphyria carriers by molecular biologic methods

Acute intermittent porphyria (AIP) is an autosomal dominant disease caused by mutations of the gene coding for hydroxymethylbilane synthase. Acute attack of AIP is a potentially life-threatening condition precipitated by certain drugs, alcohol, fasting and stress. Biochemical diagnosis before the manifestation of the symptoms is problematic, and genetic screening is required to identify asymptotic carriers. The aim of this study was to establish a fast, reproducible and reliable genetic method to detect mutations causing AIP. Exon 10 of one healthy individual and 12 AIP patients was studied using a recently developed method, temporal temperature gradient electrophoresis (TTGE). Mutation of exon 10 was detected using TTGE in one patient, DNA sequence analysis confirmed the presence of a heterozygous point mutation causing substitution of the arginine in position 173 of the gene with glutamine. These results were also confirmed using restriction enzyme analysis, and this method and TTGE identified a child of this patient as an asymptotic carrier of AIP.