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.     

Comparison of complementary and genomic DNA sequencing for the detection of mutations in the HMBS gene in British patients with acute intermittent porphyria: identification of 25 novel mutations

Acute intermittent porphyria (AIP) is a low-penetrant autosomal dominant disorder caused by mutations in the hydroxymethylbilane synthase (HMBS) gene. Direct detection of mutations is becoming the method of choice for the accurate identification of asymptomatic affected individuals within AIP families so that they can be advised to avoid drugs and other compounds that provoke the life-threatening acute neurovisceral crises that characterise the condition. We describe a prospective comparison of direct automated sequencing of cDNA (29 patients) or genomic DNA (28 patients) to identify HMBS mutations in 57 patients referred consecutively for mutational analysis; 39 different mutations were identified in 54 patients. The sensitivity of the cDNA and genomic DNA methods was 69% and 95%, respectively, indicating that analysis of genomic DNA provides a higher mutation detection rate. Thirty mutations were restricted to a single family; only one (R173W) occurred in more than three families. Of the mutations (6 missense, 8 splice defects, 10 frameshifts, 1 nonsense), 25 have not been reported previously. One novel mutation (344+33G→T) was located in a putative intron splice enhancer in intron 7. Our results define the extent of allelic heterogeneity and the types (41% missense; 59% truncating) and distribution (35% in exons 10, 12, 14) of HMBS mutations, for AIP in the United Kingdom. Received: 4 January 1999 / Accepted: 19 March 1999     

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.     

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.     

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.     

Genetic investigation of the porphobilinogen deaminase gene in Swedish acute intermittent porphyria families

A total of 12 mutations associated with acute intermittent porphyria (AIP) have been detected in the porphobilinogen deaminase gene in Swedish AIP families. Three of them are newly discovered and unique to the Swedish population: a splice mutation in intron 6 (int6+1), a missense mutation in exon 11 (Gly216Asp) and a TG deletion in exon 14. Received: 23 December 1996 / Accepted: 17 February 1997     

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.     

A novel point mutation in an acceptor splice site of intron 32 (IVS32 A–12→G) but no exon 3 mutations in the glycogen debranching enzyme gene in a homozygous patient with glycogen storage disease type IIIb

Genetic deficiency of the glycogen-debranching enzyme (debrancher) causes glycogen storage disease type III (GSD III), which is divided into two subtypes: IIIa and IIIb. In GSD IIIb, glycogen accumulates only in the liver, whereas both liver and muscles are involved in GSD IIIa. The molecular basis for the differences between the two subtypes has not been fully elucidated. Recently, mutations in exon 3 of the debrancher gene were reported to be specifically associated with GSD IIIb. However, we describe a homozygous GSD IIIb patient without mutations in exon 3. Analysis of the patient’s debrancher cDNA revealed an 11-bp insertion in the normal sequence. An A to G transition at position –12 upstream of the 3′ splice site of intron 32 (IVS 32 A^–12→G) was identified in the patient’s debrancher gene. No mutations were found in exon 3. Mutational analysis of the family showed the patient to be homozygous for this novel mutation as well as three polymorphic markers. Furthermore, the mother was heterozygous and the parents were first cousins. The acceptor splice site mutation created a new 3′ splice site and resulted in insertion of an 11-bp intron sequence between exon 32 and exon 33 in the patient’s debrancher mRNA. The predicted mutant enzyme was truncated by 112 amino acids as a result of premature termination. These findings suggested that a novel IVS 32 A^–12→G mutation caused GSD IIIb in this patient. Received: 1 August 1997 / Accepted: 22 September 1997     

Mutations of the ATM gene detected in Japanese ataxia-telangiectasia patients: possible preponderance of the two founder mutations 4612del165 and 7883del5

The ATM (A-T, mutated) gene on human chromosome 11q22.3 has recently been identified as the gene responsible for the human recessive disease ataxia-telangiectasia (A-T). In order to define the types of disease-causing ATM mutations in Japanese A-T patients as well as to look for possible mutational hotspots, reverse-transcribed RNA derived from ten patients belonging to eight unrelated Japanese A-T families was analyzed for mutations by the restriction endonuclease fingerprinting method. As has been reported by others, mutations that lead to exon skipping or premature protein truncation were also predominant in our mutants. Six different mutations were identified on 12 of the 16 alleles examined. Four were deletions involving a loss of a single exon: exon 7, exon 16, exon 33 or exon 35. The others were minute deletions, 4649delA in exon 33 and 7883del5 in exon 55. The mutations 4612del165 and 7883del5 were found in more than two unrelated families; 44% (7 of 16) of the mutant alleles had one of the two mutations. The 4612del165 mutations in three different families were all ascribed to the same T→A substitution at the splice donor site in intron 33. Microsatellite genotyping around the ATM locus also indicated that a common haplotype was shared by the mutant alleles in both mutations. This suggests that these two founder mutations may be predominant among Japanese ATM mutant alleles. Received: 15 September 1997 / Accepted: 12 January 1998     

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.     

Novel compound heterozygous mutations for lipoprotein lipase deficiency. A G-to-T transversion at the first position of exon 5 causing G154V missense mutation and a 5' splice site mutation of intron 8.

We systematically investigated the molecular defects causing a primary LPL deficiency in a Japanese male infant (patient DI) with fasting hyperchylomicronemia (type I hyperlipoproteinemia) and in his parents. Patient DI had neither LPL activity nor immunoreactive LPL mass in the pre- and post-heparin plasma. The patient was a compound heterozygote for novel mutations consisting of a G-to-T transversion at the first nucleotide of exon 5 [+1 position of 3' acceptor splice site (3'-ass) of intron 4] and a T-to-C transition in the invariant GT at position +2 of the 5' donor splice site (5'-dss) of intron 8 (Int8/5'-dss/t(+2)c). The G-to-T transversion, although affecting the 11 nucleotide of the 3'-consensus acceptor splice site, resulted in a substitution of Gly(154) to Val (G154V; GG(716)C(-->)GTC). The mutant G154V LPL expressed in COS-1 cells was catalytically inactive and hardly released from the cells by heparin. The Int8/5'-dss/t(+2)c mutation inactivated the authentic 5' splice site of intron 8 and led to the utilization of a cryptic 5'-dss in exon 8 as an alternative splice site 133 basepairs upstream from the authentic splice site, thereby causing joining of a part of exon 8 to exon 9 with skipping of a 134-bp fragment of exon 8 and intron 8. These additional mutations in the consensus sequences of the 3' and 5' splice sites might be useful for better understanding the factors that are involved in splice site selection in vivo.     

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.