The biochemical and molecular spectrum of ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTCase) deficiency, the most common inherited urea cycle disorder, is transmitted as an X-linked trait. The clinical phenotype in affected males as well as heterozygous females shows a spectrum of severity ranging from neonatal hyperammonaemic coma to asymptomatic adults. The ornithine transcarbamylase enzyme is a trimer with three active sites per holoenzyme molecule, each of which is composed of an interdomain region of one polypeptide and a polar domain of the adjacent polypeptide. The OTC gene is located on the short arm of the X-chromosome and one of the two alleles undergoes inactivation in female cells. Approximately 140 mutations have been found in families affected with OTCase deficiency, most having their own 'private' mutation. Large deletions of one exon or more are seen in approximately 7% of patients, small deletions or insertions are seen in about 9%, and the remaining mutations are single base substitutions. Approximately 15% of mutations affect RNA splicing sites. The recurrent mutations are distributed equally among CpG dinucleotide hot spots. Generally, mutations causing neonatal disease affect amino acid residues that are 'buried' in the interior of the enzyme, especially around the active site, while those associated with late onset and milder phenotypes tend to be located on the surface of the protein. Very few mutations have been found in the sequence of the leader peptide, proportionally much fewer than in the sequence of the mature enzyme. Only few of the mutations have been expressed in bacteria or mammalian cells for the study of their deleterious mechanisms. Examples of expressed mutations include R277W and R277Q associated with late-onset disease, which markedly increase the Km for ornithine, shift the pH optimum to more alkaline and decrease the thermal stability of the purified mutant enzyme. R141Q (neonatal disease) disrupts the active site, whereas the purified R40H mutant has normal catalytic function and this mutation is likely to affect posttranslational processing such as mitochondrial targeting. It appears that most new mutations occur in male sperm and are then passed on to a transmitting heterozygous female. Uncommonly, mild mutations are transmitted by asymptomatic males to their daughters, subsequently resulting in clinical disease of males in future generations. The causes for variable expressivity of these mutations are currently unknown but are likely to involve a combination of environmental and genetic modifiers.

Polymorphisms in the human ornithine transcarbamylase gene useful for allele tracking. Mutations in brief no. 193. Online.

Sequences of amplified DNA fragments from the ornithine transcarbamylase (OTC) genes were screened for 10 polymorphic single base substitutions in order to determine their frequency in the population. Six of the polymorphisms showed a frequency of 0.02 or less. The remaining four polymorphisms showed a range in frequency from 0.125 to 0.5. Based on the allele frequencies of the four common polymorphisms, we calculated that a female has an 87% chance of being heterozygous for at least one locus. These intragenic sequence variations can be used for tracking of deleterious OTC alleles whenever the deleterious mutation cannot be found and should also prove useful for prenatal diagnosis.

Identification of 'private' mutations in patients with ornithine transcarbamylase deficiency.

The majority of cases of ornithine transcarbamylase deficiency are due to novel mutations making it impossible to develop common methods for genetic analysis. However, identification of causative mutations has important implications for diagnosis (particularly prenatal diagnosis), prediction of likely course and outcome and the eventual possibility of gene therapy. As part of a continuing study of ornithine transcarbamylase deficiency, we now report an additional thirty novel mutations in the ornithine transcarbamylase gene, together with a brief summary of their clinical presentations.

Clinical and biochemical heterogeneity in females of a large pedigree with ornithine transcarbamylase deficiency due to the R141Q mutation.

A large family with ornithine transcarbamylase deficiency due to mutation R141Q was ascertained through a propositus who presented with acute neonatal hyperammonemic coma. Of 13 females at risk, 11 were evaluated clinically and had laboratory studies performed. Seven were found to be heterozygous for the mutation. Of these seven, five had chronic clinical symptoms and two were asymptomatic. None of the heterozygotes had elevated plasma ammonia on random testing. Of the five symptomatic females, three had markedly elevated plasma glutamine levels on random testing, while two had levels in the upper range of normal. Plasma citrulline and arginine levels were somewhat lower in the symptomatic individuals but still within the normal range. Five heterozygotes who were tested had either spontaneous orotic aciduria or elevated orotic acid following ingestion of allopurinol, whereas one unaffected female and one unaffected male had normal allopurinol tests. A higher than expected proportion of female heterozygous for the R141Q mutation were clinically and biochemically symptomatic but remained undiagnosed for many years. Plasma glutamine determination and allopurinol testing should be performed in females who present with a combination of relatively non-specific symptoms detailed in this report.

Relative frequency of mutations causing ornithine transcarbamylase deficiency in 78 families.

Approximately 90 different mutations associated with ornithine transcarbamylase (OTC) deficiency are currently known. Thus, the majority represent private mutations. However, some of the mutations seemed to be recurrent. Our laboratories identified apparent deleterious mutations in 78 consecutive families with OTC deficiency by screening all exons and exon/intron borders using single-strand conformational polymorphism (75 families) or sequencing of the entire coding sequence (3 families). Large deletions of one or more exons were found in 8% of families and approximately 10% had small deletions or insertions of 1-5 bases. Splice site mutations were found in 18% of families. Contrary to previous reports, recurrent point mutations seemed to be equally distributed among most CpG dinucleotides rather than show prevalent mutations. No single point mutation had a relative frequency of more than 6.4%. Of the 64 families with nucleotide substitution, 24 (38%) were G to A with the next most common being C to T (16%) and A to T (11%).

Mutations and polymorphisms in the human ornithine transcarbamylase gene: mutation update addendum.

This mutation update addendum summarizes 30 new mutations and polymorphisms found in the ornithine transcarbamylase (OTC) gene since the publication in this journal of the first mutation update. Thus, more than 60 mutations and polymorphisms in the OTC gene are currently known. Most of the mutations have been seen in a single family and the few recurrent mutations occurred in CpG dinucleotides. The presumed deleterious effects of most mutations await confirmation by appropriate expression studies. Once the tertiary structure of the enzyme is fully known, and the functional domains established, the effects of mutations, or lack thereof, could be better predicted.

Four new mutations in the ornithine transcarbamylase gene.

We characterized four new mutations in the ornithine transcarbamylase (OTC) gene in three male infants who died from acute neonatal hyperammonemia and one male infant with late onset disease. OTC enzymatic activity was undetectable in the livers of the three neonates, whereas residual enzymatic activity was present in the fourth patient. All 10 exons of the OTC gene were amplified by the polymerase chain reaction (PCR) from genomic DNA of the four patients. The amplified DNA was screened for abnormal gel electrophoretic migration patterns via single-strand conformational polymorphism (SSCP). One patient showed an abnormal SSCP pattern of exon 8 and exon 9, a second patient had an abnormal exon 6, a third had an abnormal exon 9, and the fourth patient revealed an abnormal exon 3. Sequencing of the abnormal exons revealed that the first patient had a deleterious mutation in exon 9 consisting of a G-->T transversion in codon 310 causing a Glu-->stop coding change. The abnormal exon 8 of this patient contained a common polymorphism consisting of an A-->G transition in codon 270 resulting in Gln-->Arg code change. The abnormal exon 6 of the second patient contained an A-->G transition in codon 183 causing a Tyr-->Cys change. Exon 9 of the third patient contained a deletion of a thymine nucleotide (base 882) resulting in a shift of the reading frame and a code for premature termination 28 codons downstream. The fourth patient with a "milder" clinical presentation had an A-->T transversion in exon 3 (codon 88) causing a Lys-->Asn change.