Biomedical subjects
Publications and source records attributed to R G Cotton.
The 6th International HUGO Mutation Database Meeting was held on March 27, 1999, in conjunction with the Human Genome Meeting HGM 1999, at the Brisbane Conference and Exhibition Centre, Brisbane, Australia. Meeting highlights are described.
The Fifth International Mutation Detection Workshop brought together inventors and major users of mutation detection methodology in a freshly refurbished 17(th) century monastery in northern Italy. There were over 120 registrants from 22 nations, all of which gave either a poster or oral presentation, making it difficult to distill the meeting into a few pages. Here we review the meeting by method type and describe highlights within each. It was clear, however, that with the imminent completion of the Human Genome Project and the recent emphasis on the utility of single nucleotide polymorphisms (SNPs), many presenters emphasized the high-throughput aspect of their methods as well as cost.
Mutation detection techniques are often limited by sensitivity, ease of use and short fragment lengths. Enzyme mismatch cleavage (EMC) is a technique capable of rapidly scanning 1 kbp fragments of DNA for mutations. It relies on the ability of a bacteriophage resolvase enzyme, T4 endonuclease VII, to cleave DNA at single base pair mismatches and small heteroduplex loops. Originally the process was performed using radioactively labeled DNA and the results analysed after denaturing polyacrylamide gel electrophoresis and autoradiography. However, access to systems capable of detecting fluorescent species migrating through a gel and the widespread availability of fluorescently tagged primers have greatly improved upon the original technique. A number of mutations were detected using fluorescent EMC and the results compared to performing the technique using radiolabeled DNA. Fluorescent EMC detected the presence, position and number of mutations in DNA fragments as large as 1 kbp. The fluorescent method was found to have advantages over the original method in its ease of use, increase in signal-to-noise ratio and the ability to multiplex samples by labeling DNA fragments with different fluorophores. This improvement on an already established method provides a sensitive, robust technique for mutation detection.
Detection and amplification of mutations in genes in a cheap, 100% effective manner is a major objective in modern molecular genetics. This ideal is some way away and many methods are used each of which have their own particular advantages and disadvantages. Sequencing is often thought of as the 'gold standard' for mutation detection. This perception is distorted due to the fact that this is the ONLY method of mutation identification but this does not mean it is the best for mutation detection. The fact that many scanning methods detect 5-10% of mutant molecules in a wild type environment immediately indicates these methods are advantageous over sequencing. One such method, the Chemical Cleavage method, is able to cut the costs of detecting a mutation on order of magnitude and guarantees mutation detection as evidenced by track record and the fact that each mutation has two chances of being detected.
Phenylalanine hydroxylase converts phenylalanine to tyrosine, a rate-limiting step in phenylalanine catabolism and protein and neurotransmitter biosynthesis. It is tightly regulated by the substrates phenylalanine and tetrahydrobiopterin and by phosphorylation. We present the crystal structures of dephosphorylated and phosphorylated forms of a dimeric enzyme with catalytic and regulatory properties of the wild-type protein. The structures reveal a catalytic domain flexibly linked to a regulatory domain. The latter consists of an N-terminal autoregulatory sequence (containing Ser 16, which is the site of phosphorylation) that extends over the active site pocket, and an alpha-beta sandwich core that is, unexpectedly, structurally related to both pterin dehydratase and the regulatory domains of metabolic enzymes. Phosphorylation has no major structural effects in the absence of phenylalanine, suggesting that phenylalanine and phosphorylation act in concert to activate the enzyme through a combination of intrasteric and possibly allosteric mechanisms.
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Previous studies have shown that genotype correlates with biochemical phenotype in treated phenylketonuria. If there is a strong correlation between genotype and intellectual phenotype of untreated patients, it would be possible to determine which individuals would have normal intelligence without treatment. In this study, 42 families with untreated phenylketonuria were analyzed to examine whether there was an association between genotype and untreated intellectual phenotype. Previously 12 of the 42 families were genotyped; now the genotyping of these patients is almost complete (40/42), a more thorough investigation was possible. Although the predicted phenylalanine hydroxylase (PAH) enzyme activity, based on genotype, showed an association with the patients' intellectual phenotype, the extensive overlap between the groups means the association is of little clinical value. Unrelated individuals with the same genotype and also siblings were found to have very different intellectual phenotypes. These phenotypic differences could not be explained by a difference in diet; therefore, we propose that another gene or genes may be modifying the intellectual phenotype of untreated patients. A preliminary search for possible modifying genes was performed. The possibility that a modifying gene was linked to the PAH gene on chromosome 12 was investigated using markers closely linked to the gene; however, no evidence for a modifying gene close to the PAH gene was found. Tyrosine hydroxylase was chosen as a candidate gene, because it can perform the same reaction as PAH. Using a common polymorphism within the gene, we found that this gene did not cause the discordant results and thus, did not modify the PAH phenotype.
Phenylalanine hydroxylase (PAH) exists as an equilibrium of dimers and tetramers. However, there is little information concerning the inter- or intra-molecular interactions required for enzyme quaternary structure. It is predicted that the formation of a PAH tetramer will require at least two points of contact per enzyme subunit. Sequence analysis has suggested the existence of a C-terminal domain with characteristics of a leucine zipper or a variant of this called a coiled-coil. By deletion of 24 amino acids from the C-terminus or conversion of leucine 448 to an alanine residue, we have shown that this putative leucine zipper/coiled-coil domain is involved in the assembly of an active enzyme tetramer from dimers. The removal of this C-terminal domain of PAH reduces enzyme activity but does not abolish it. Furthermore, we report that an alanine 447 to aspartate mutation associated with phenylketonuria may affect subunit assembly which suggests the formation of enzyme tetramers is physiologically relevant. Our analysis of subunit interactions in vivo, show that in the absence of the C-terminal coiled-coil domain, dimers can form and this is only possible when the N-terminal domain is present. This provides the first evidence that N-terminal domain is required for multimerisation. We propose that the N-terminal regulatory domain in conjunction with the C-terminal coiled-coil domain, mediates the formation of fully active enzyme tetramers.
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Chemical cleavage of mismatch (CCM), also known as chemical mismatch cleavage (CMC) or the HOT (hydroxylamine/osmium tetroxide) chemical method, has been used for detection of sequence variability with many systems since it was first described. Recently, adaptation to fluorescence-based detection systems has fundamentally changed both the execution and analysis of CCM. This review will outline major advances in the methodology of CCM, from the advent of PCR through fluorescent analysis, and includes applications and modifications of CCM.
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Dihydropteridine reductase (DHPR) is an enzyme involved in recycling of tetrahydrobiopterin (BH4), the cofactor of the aromatic amino acid hydroxylases. Its deficiency is characterized by hyperphenylalaninemia due to the secondary defect of phenylalanine hydroxylase and depletion of the neurotransmitters dopamine and serotonin, whose syntheses are controlled by tryptophan and tyrosine hydroxylases. The DHPR cDNA has been cloned and mapped on 4p15.3. In the present study we report the genomic structure of the DHPR gene (QDPR). This gene includes seven exons within a range of 84-564 bp; the corresponding introns are flanked by canonic splice junctions. We also present a panel of PCR primers complementary to intronic sequences that greatly facilitates amplification of the gene and provides a genomic DNA approach for mutation detection. We have used this approach to study six patients with DHPR deficiency. Four known mutations (G23D, H158Y, IVS5G+ 1A, R221X) and two new mutations (Y150C and G218ins9bp) were found. The Y150C mutation was found in compound heterozygosity with G23D, a mutation always associated with a severe phenotype in homozygous patients. This patient has an intermediate phenotype (good response to monotherapy with BH4). The mutant enzyme for Y150C was expressed in an E. coli system. Comparison of its kinetic parameters with those of the G23D mutant enzyme showed that it is not as effective as the wild-type enzyme, but is more active than the G23D mutant. This patient's intermediate phenotype is thus due to the mild DHPR mutation Y150C. Correlations between genotypes and phenotypes were also found for the other mutations.
Detection of mutations in genes is vital throughout biology, however, this activity is time-consuming, expensive and requires a high degree of skill. This is unsatisfactory in a field which is increasing importance. Around 10-12 methods are commonly used with some predominating. All have their advantages and disadvantages and none is perfect. Sequencing is said to be the gold standard for detecting new mutations (and must be used to define it), but six or so methods have been described to make the search quicker to avoid sequencing the whole gene. These are called scanning methods. Other methods are used to detect known mutations and referred to as diagnostic methods. These methods will be briefly reviewed. Once mutations are described, they are usually published. The mutation lists are collected for convenience, analysis or research. In recent years, it has been realised that these lists are vital for research, patient care and commercial activities. These activities will be reviewed and the co-ordinating role of the HUGO Mutation Database Initiative will be outlined.
Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.
Mutations in the dihydropteridine reductase (DHPR) gene result in hyperphenylalaninaemia and deficiency of various neurotransmitters in the central nervous system, causing severe neurological symptoms. We studied two Japanese patients with DHPR deficiency and identified a missense and a splicing error mutation, respectively. A homozygous missense mutation (tryptophan36-to-arginine) was detected in patient 1. The mutation abolished DHPR activity according to in vitro expression studies. The DHPR mRNA in patient 2 was markedly decreased. Reverse transcription-polymerase chain reaction of the mRNA generated a cDNA fragment with a 152-bp insertion. The inserted sequence contained a termination codon, which was likely to affect the stability of the mRNA. Analysis of genomic DNA showed that the insertion was derived from putative intron 3 of the DHPR gene, and an intronic A-to-G substitution was present adjacent to the 3'-end of the inserted sequence. The nucleotide change generated a sequence similar to an RNA splice donor site and probably activated an upstream cryptic acceptor site, thus producing an abnormal extra exon.
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The enzyme mismatch cleavage (EMC) method relies on the use of the resolvase T4 Endonuclease VII to cleave and thus detect mismatches in heteroduplex DNA formed by annealing normal DNA with mutant DNA. Detection is based on cleavage 3' to the mismatch within a few nucleotides. We report the detection of all 81 different homozygous single-basepair changes tested and present in the mouse beta-globin promoter by using the EMC method with a single set of conditions. Efficiency of cleavage was rated as strong, medium, or weak based on the intensity of the cleavage product(s) compared with background bands on autoradiography. We expect this method to detect near 100% of mutations.