Acute intermittent porphyria (AIP) is an inborn error of metabolism which is caused by deleterious mutations in the hydroxymethylbilane synthase (HMB-S) gene. The overall aims of the present study were to search for and characterise mutations in the HMB-S gene and to develop an in vitro expression system to confirm the pathogenic nature of these mutations. Thirty individuals from fifteen families with AIP from South Africa, Scotland, England and the Republic of Ireland were studied. Initially, the entire coding sequence of the HMB- S gene was screened using heteroduplex analysis (HA) and chemical cleavage mismatch (CCM) analysis. Any differences in the band patterns, when compared to the negative control in both techniques, were characterised by direct sequencing. The same region of the genomic DNA was also sequenced. This step was especially important in characterising splice site mutations responsible for exon skipping. Using the above strategies, six missense mutations, R22C, R26C, R26H, R116W, R173Q and L177R, one splice site mutation (345-1G?A) leading to the skipping of exon 8 and two frameshift mutations (771insT and del1002/1003/1004) were identified. Four of these mutations, R22C, 345-1G?A, 771insT and del1002/1003/1004, are novel. All the missense mutations resulted in the substitution of highly conserved residues, hence are considered to be potentially pathogenic. In addition, alternative splicing of exons 3 and 12, resulting in their exclusion from a proportion of mRNA transcripts, was identified in thirty AIP patients and forty-four healthy individuals. The R22C mutation (R7 in the E. coli model) in exon 3 affects the highly conserved Arg22 residue and is caused by a C to T substitution at nucleotide 64. Arginine, a basic residue, is substituted by cysteine, a neutral polar amino acid and this is predicted to destabilise the protein. This mutation, initially detected in two Scottish siblings, was subsequently identified in three other family members by employing a simple restriction enzyme analysis using Hinf I. R26C (R11 in E. coli) is the result of a C to T transition in exon 3 at position 76. The highly conserved Arg26 residue in twelve species is substituted by the polar neutral cysteine residue. This mutation was present in two Irish siblings and was identified in three further relatives by performing a simple Aci I restriction enzyme analysis. R26H is caused by a G to A transition of the adjacent base following that causing R26C and was confirmed by an Aci I digestion. R116W (R101 in E. coli) in exon 8 was identified in three unrelated South African patients and raised the possibility of these patients having a common Dutch ancestry, due to the high prevalence of this mutation in the Dutch population. R116W is a consequence of a C to T transition at nucleotide 346 which was confirmed by an Aci I digestion. A destabilisation of the protein is expected with the substitution of the basic amino acid arginine with tryptophan, a neutral non-polar amino acid. The mutation in exon 10, R173Q (R155 in E. coli), is caused by a G to A transition at position 518 and a loss of an Nci I recognition site indicates a base change. In this case, the basic residue arginine is substituted with glutamine, a polar neutral residue. This replacement inhibits substrate binding and chain elongation. L177R (LI59 in E. coli), in exon 10, is the result of a T to G transversion at nucleotide 530, causing the non-polar neutral residue leucine to be replaced by the basic amino acid arginine. This introduces a destabilising effect in the mature protein and impairs its normal function. The Aci I restriction enzyme was employed to identify this mutation in five members of a Scottish family. 345-1G?A is a 3' acceptor splice site mutation at position -1 of intron 7 which causes the skipping of exon 8 in three related individuals from England. 26 amino acids are predicted to be absent from the protein product but the translational reading frame is not altered. However, the codon at the junctions of exons 7 and 8 of the normal mRNA is now altered and encodes for asparagine instead of lysine. The application of Eco RII confirmed this mutation. 771insT was identified in exon 12 of a South African patient which results in a shift in the reading frame and the alteration of the thirty-two amino acids following this insertion before reaching a premature termination of translation at codon 290. This mutation affected a Sty I recognition site, which was used for confirmation. A protein product containing 289 amino acids is expected instead of the 361 amino acids normally found in a healthy individual, equivalent to the loss of approximately 20% of the protein. The mutation, delG1002/1003/1004, also results in a termination codon, this time at codon 343, and consequently a protein containing 342 amino acids is expected. This corresponds to a loss of approximately 5% of the mature protein. Five of the missense mutations, R22C, R26C, R26H, R116W and R173Q, occur at the hypermutable CpG dinucleotide, a hotspot for mutations. The normal occurrence of alternative splicing of exons 3 and 12 had not been previously reported.(Abstract shortened by ProQuest.).
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