Ornithine transcarbamylase deficiency resulting from a C-to-T substitution in exon 5 of the ornithine transcarbamylase gene.

To define the molecular basis for the TaqI site alteration in the ornithine transcarbamylase (OTC) (E.C.2.1.3.3) gene of a female patient with mild OTC deficiency, we used a combination of genomic amplification followed by direct sequencing and oligodeoxyribonucleotide hybridization. We obtained evidence for a C-to-T substitution in exon 5 (codon 141) of this gene. This mutation generates a stop codon, in place of Arg, at amino acid 109 of the mature OTC protein. The mutation arose, de novo, in a germ cell of one of the parents.     

New mutation and prenatal diagnosis in ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) (E.C.2.1.3.3) is an X-linked hepatic enzyme in the urea cycle necessary for ammonia detoxification. Deficiency of OTC results in neonatal hyperammonemia, coma, and death in childhood. Because fibroblasts do not express OTC, prenatal diagnosis in the past has required fetal liver biopsy. Using a complementary DNA (cDNA) for OTC for Southern blot analysis of genomic DNA, we have found probands with complete OTC deficiency from two unrelated families in whom the same TaqI restriction endonuclease site has been altered because of independent, but not necessarily identical, mutations in the OTC gene, suggesting that this site may be a relative hotspot for mutation at a location that is critical for normal gene function. This TaqI alteration has allowed the identification of the individual in each family in whom the mutation originated as well as the exclusion of a recurrence of OTC deficiency in a male fetus at risk for the disease. OTC deficiency joins the growing list of genetic disorders for which Southern blot analysis allows accurate heterozygote detection and prenatal diagnosis in conditions for which they were not previously available.     

The R40H mutation in a late onset type of human ornithine transcarbamylase deficiency in male patients

Ornithine transcarbamylase (OTC) deficiency is an X-linked trait and is one of the most frequent of the inherited urea cycle enzyme deficiencies. Most male patients with OTC deficiency develop a hyperammonemic crisis and die in the neonatal period or in early infancy. In contrast to those patients, in some male patients the disease first becomes overt in adolescence or during the reproductive age period. In the present report, we describe six such male patients who first developed clinical signs at ages ranging from 6 to 58 years, all of whom came from a limited area of the northern part of Kyushu Island in southern Japan. The mutation analysis disclosed a R40H mutation in exon 2 of the OTC gene in each of these patients. Transmission of this mutant gene through paternal lineage as well as through maternal lineage was documented in one family. The levels of mRNA of the mutant OTC gene expressed in transfected Cos 1 cells and in the liver tissue obtained by biopsy in one patient were both similar to those of the wild-type gene. The activity of the mutant OTC was, however, decreased to a level of 28% of the wild-type OTC, and the levels of the mutant OTC protein expressed in Cos 1 cells were decreased, as assessed by western blot analysis. Apparent K _m values of the mutant enzyme for ornithine (1.1 mM) and carbamylophosphate (2.0 mM) were similar to those of the wild-type enzyme. Both enzymes gave similar pH-dependency profiles, giving a maximal activity at pH 7.8–7.9. Activity of wild-type OTC expressed in Cos 1 cells did not change after five cycles of freezing and thawing, whereas that of the mutant OTC decreased to 17% by this treatment. These results suggest that deficiency is due to inactivation of the mutant OTC under certain conditions. Received: 15 May 1996     

Improved molecular diagnostics for ornithine transcarbamylase deficiency.

Since the cloning of the cDNA for X-linked ornithine transcarbamylase (OTC) in 1984, diagnostic accuracy of OTC deficiency for prenatal and carrier detection has been greatly improved by the use of linkage analysis. However, the use of RFLP-based diagnosis is limited in this and in other new mutation diseases. Here we report both the use of direct mutation detection by new PCR-based techniques and our experience with linkage-based diagnosis in 18 families. We have previously reported the use of chemical mismatch cleavage to detect mutations first in amplified mRNA and then in genomic DNA of patients. This technique has now been utilized for prenatal diagnosis. Primers for specific amplification of OTC exons 1, 3, 5, 9, and 10 have been developed and been employed to map deletions of the OTC gene in two families. These primers also have been used to detect alterations in the TaqI sites found in exons 1, 3, 5, and 9. Four novel mutations of the OTC gene leading to abolition of a TaqI site in the OTC cDNA were discovered. One of these mutations is in exon 1; two lie in exon 3; and one is in exon 9. In addition, we have used the PCR products as probes to identify the exon-specific bands seen on Southern blots and to map the polymorphic BamHI and MspI sites, which are commonly used for linkage analysis. This information will facilitate the interpretation of altered band patterns seen in deletion cases and in cases of point mutations affecting restriction sites. Utilization of the appropriate combination of these molecular techniques permitted accurate diagnostic evaluations in 17 of 18 families.     

Single-strand conformational polymorphism and direct sequencing applied to carrier testing in families with ornithine transcarbamylase deficiency

Single-strand conformational polymorphism (SSCP) and direct sequencing were used to confirm or deny carrier status in three families with ornithine transcarbamylase (OTC) enzyme deficiency. Two male probands with late onset OTC deficiency, whose private mutations were previously characterized, inherited the mutations form their heterozygous mothers. One of the heterozygous mothers had a false negative allopurinol test. Three female siblings of the two male probands were tested, one proved to be a carrier of the respective mutation while the other two were found to have normal alleles. In the third family, the proband was a female with late onset presentation of OTC deficiency. We found a new point mutation in this girl consisting of a guanine-tocytosine transversion at nucleotide 520 resulting in a substitution of proline for alanine at amino acid 142 of the mature OTC protein. We confirmed that this mutation occurred spontaneously and that neither of the two parents carries this mutation. We conclude that SSCP, in conjunction with direct sequencing, is a useful technique that can be practically applied for carrier testing in families with OTC deficiency.     

Screening for gene deletions and known mutations in 13 patients with ornithine transcarbamylase deficiency.

We analyzed DNA from 13 males with ornithine transcarbamylase (OTC) deficiency for gene deletions and known point mutations using the polymerase chain reaction (PCR), allelle-specific oligonucleotide (ASO) hybridization, and Southern blotting with full-length OTC cDNA and exon-specific probes. Three patients were found to have deletions: one was missing the whole OTC gene; a second patient had a deletion of both exon 7 and 8; and the third had a deletion of exon 9. Only one of the remaining 10 patients had a known point mutation consisting of a G-to-A change in nucleotide 422 of the sense strand resulting in a glutamine substitution for arginine at amino acid 109 of the mature OTC protein. This study describes the integration of various molecular methods to screen OTC-deficient patients for deletions and points mutations. Two new deletions within the OTC gene are described.     

Demonstration of the spf-ash mutation in Spanish patients with ornithine transcarbamylase deficiency of moderate severity

We have found in patients with ornithine transcarbamylase (OTC) deficiency from two Spanish families (A and B), replacement by A of G at the 3-end of exon 4 of the OTC gene. The same mutation is found in the spf-ash mouse, a rodent model of mild OTC deficiency, causing a neutral R129H mutation and inefficient splicing at the 5donor site of the exon 4-intron 4 junction, with resultant 4%–7% residual OTC activity. The mutation, detected in our patients using polymerase chain reaction (PCR) amplification of the ten OTC exons, single strand conformation polymorphism (SSCP) analysis and direct sequencing of PCR-amplified exon 4, results in the loss of a unique MspI restriction site which can be used for rapid diagnosis. The mutation was transmitted by the mother in family A and arose de novo in the patient in family B. Residual OTC activity, determined in a male and a female patient, was 1.3% and 3.5% of normal, respectively. Despite this low activity, the surviving patients have developed normally.     

Direct and indirect mutation analyses in patients with ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) is one of 5 enzymes in the detoxification of ammonia to urea, and its deficiency, an X-linked disease, is the most common inborn error of urea genesis in humans. Because of the devastating nature of the disease there is a strong demand for reliable and rapid molecular analyses in OTC families in order to offer carrier detection and prenatal diagnosis. This paper presents the efficiency of direct and indirect mutation analyses in 22 OTC families using Southern blotting and polymerase chain reaction (PCR) amplification. For 89% of the mothers with an affected child, at least 1 RFLP of the OTC locus was informative concerning prenatal diagnosis. 100% informativity was reached by using the additional flanking markers 754 and LI.28. In total, 3 deletions (14%) and 1 TaqI site mutation (4.5%) in exon 3 were detected. 13 (60%) of our 22 mothers were found to be carriers, 9 of them being obligate carriers and 4 detected by biochemical testing. 4 mothers were excluded as carriers by DNA analyses, and in 5 mothers the carrier status could not be assessed positively. DNA analyses permitted carrier detection in 32% and carrier exclusion in 55% of 22 female relatives. Prenatal diagnosis was performed in 4 families: in 1 family by direct mutation detection and in 3 families by linkage analyses. It was possible to determine the mutation origin in 6 families, all of them with male probands. In 4 families the mutation had occurred during grandpaternal spermiogenesis, suggesting higher mutation rates in males, but in 2 cases it was the result of an event during maternal oogenesis, proving that new mutations in the OTC gene do also occur in eggs. Our recommended strategy for carrier detection and prenatal diagnosis in OTC deficiency is to examine routinely Southern blots of BamHI, EcoRI, HindIII, MspI, PstI and TaqI digestions using the OTCcDNA probe pH0731 and the flanking markers 754 and LI.28, as well as the TaqI-digested PCR products of exons 3, 5 and 9.     

Identification of four novel splice site mutations in the ornithine transcarbamylase gene

Ornithine transcarbamylase (OTC) deficiency, the most common inborn error of the urea cycle, shows X-linked inheritance with frequent new mutations. Using polymerase chain reaction (PCR) amplification of the individual exons including adjacent intron sequences followed by direct sequencing of the amplimers we identified four new mutations affecting donor splice sites of introns 2, 5, 6, and 8. The mutation at the first position of intron 2 was a G to A exchange associated with acute neonatal hyperammonemia in a male patient at the age of 5 months. A G to C substitution in intron 5 was detected in a boy who developed 2 days after birth hypotonia, and respiratory distress, followed by severe hyperammonemia and terminal coma. The intron 6 mutation, a G to T substitution, was detected in a girl presenting with first episodes of vomiting and agitation at the age of 2 months. The mutation in intron 8, also a G to T transition, caused fatal hyperammonemia and early death at the age of 15 days in a male patient. We present four donor splice site mutations resulting in severe neonatal or very early onset of the disease in three boys and in one female patient. As the GT dinucleotide of the 5 donor splice site is invariant and required for correct splicing the described mutations may lead to improperly spliced mRNAs and aberrant gene products.     

Fatal hyperammonemia resulting from a C-to-T mutation at a MspI site of the ornithine transcarbamylase gene

Summary Ornithine transcarbamylase (OTC) deficiency is the most common inborn error of the urea cycle in humans and is responsible for lethal neonatal hyperammonemia in males. Partial OTC deficiency also occurs in females and can be responsible for life-threatening hyperammonemic comas in heterozygotes. The cosegregation of the trait with a 5.8-kb abnormal MspI fragment in an affected family led us to hypothesize that this unexpected migration pattern was related to the mutation event in this particular family. Using polymerase chain reaction amplification of the specific mRNA derived from a post-mortem biopsy of the liver, we found that the MspI site located in the seventh exon of the gene was abolished and we finally identified a C-to-T transition at codon 225 of the cDNA, changing a proline to a leucine in the protein. Subsequent digestion of amplified exon 7 using the restriction enzyme MspI allowed direct screening for the mutant genotype during the next pregnancy. The present study supports the view that direct detection of the mutant genotype using either Southern blotting or digestion of amplified exons of the gene can contribute to genetic counselling in non-informative families. Finally, since MspI digestions are routinely performed for restriction fragment length polymorphism-based family studies in OTC deficiency, we suggest that the possible presence of the 5.8-kb abnormal fragment should be investigated on Southern blots of affected individuals.     

Identification of RNA splicing errors resulting in human ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTC) is an X-linked, liver-specific enzyme that catalyzes the second step of the urea cycle. In humans, inherited deficiency of OTC in hemizygous affected males usually results in severe ammonia intoxication and early death. To characterize mutations responsible for OTC deficiency, we used the PCR to amplify cDNAs prepared from patient livers which demonstrated no OTC enzyme activity and no OTC cross-reacting material on western blots. In three of seven cases, smaller than normal products were observed. Sequencing of these cDNAs revealed that two were missing exon 7 of the OTC gene and that the other was missing the first 12 bp of exon 5. Sequencing of genomic DNA from these three patients revealed that one mutant missing exon 7 had a T-to-C substitution in the 5' splice donor site of intron 7. The other mutant missing exon 7 had an A-to-G change in the third position of intron 7. It is interesting that both of these mutations resulted in skipping the preceding exon rather than in inclusion of some or all of the affected intron. In the third mutant, an A-to-T substitution was found in the 3' splice acceptor site at the end of intron 4. Here, a cryptic splice acceptor site within exon 5 was used. Northern blotting of liver RNA from these patients demonstrated (a) reduced, but significant, amounts of OTC mRNA in one of the patients who had a deleted exon 7 but (b) very little OTC mRNA in the other two patients. We propose that these point mutations, which result in aberrant splicing of the OTC pre-mRNAs, lead to OTC deficiency through either decreased efficiency of mRNA export from the nucleus to the cytosol or synthesis of enzyme subunits that are unstable and rapidly degraded. We speculate that abnormal mRNA splicing may represent a relatively common mechanism in the pathogenesis of this disease.     

Expression of four mutant human ornithine transcarbamylase genes in cultured Cos 1 cells relates to clinical phenotypes

Ornithine transcarbamylase (OTC) deficiency is an X-linked disease with a heterogeneous phenotype, even in affected males. To detect mutations in the OTC gene using genomic DNA, we have developed a method in which all exons and adjacent introns are amplified and sequenced. Although this approach detected mutations in many cases, the relationship between a mutation and the OTC phenotype was not firmly established. Therefore, we investigated the issue by expression analysis of mutant OTC cDNA in cultured cells. Four mutant OTC cDNAs were constructed, based on the reported cases, using our newly developed method. The normal (wild-type) human OTC cDNA was reproducibly expressed at high levels in these Cos 1 cells. Predicted OTC activities of mutant OTC cDNAs ranged from 0% to 8.9% of the normal level together with variable amounts of the enzyme protein. The predicted enzyme activities account for the clinical phenotype of the disease. Our observations confirm that these mutations are responsible for OTC deficiency in these patients.     

Prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from maternal blood

We have developed a method that allows the prenatal DNA diagnosis of ornithine transcarbamylase (OTC) deficiency by using a single fetal nucleated erythrocyte (NRBC) isolated from maternal blood. OTC gene analysis of a male patient (TF) with early onset OTC deficiency was performed by single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. To investigate the possible prenatal diagnosis of OTC deficiency, maternal blood was obtained at 13 weeks of gestation of a subsequent pregnancy, from the mother of patient TF. NRBCs in the maternal blood were separated by using the density gradient method and then collected with a micromanipulator. The entire genome of a single NRBC was amplified by primer extension preamplification (PEP). The human leukocyte antigen (HLA)-DQ alpha genotype and sex were determined from small aliquots of the PEP product. The HLA-DQ alpha genotype of each of the parents of the male patient was also determined. Once a single NRBC had been identified as being of fetal origin, the OTC gene was analyzed by using the restriction fragment length polymorphism (RFLP) method. DNA analysis revealed a point mutation in exon 9 of the OTC gene in the OTC-deficient patient (TF). All NRBCs retrieved from maternal blood were successfully identified as being of fetal origin by HLA-DQ alpha genotyping and sex determination. RFLP analysis demonstrated that the fetal OTC gene was normal. This is the first study to successfully diagnose OTC deficiency prenatally, by using a single fetal NRBC from the maternal circulation. Such prenatal DNA diagnosis is non-invasive and can be applied to other genetic diseases, including autosomal and X-linked diseases. Received: 19 December 1997 / Accepted: 14 February 1998     

Genetic control of ornithine transcarbamylase induction in chick kidney

After ornithine transcarbamylase (OTC) induction by egg-yolk feeding, OTC activity increases rapidly in chicks bearing an Ocb gene. This response to an egg yolk diet does not appear in chicks having no Ocb gene (showing low OTC activity). The chicks showing intermediate OTC activity also respond to the diet, but moderately. Crossing experiments revealed that OTC induction by egg yolk-diet feeding is inherited as a simple autosomal dominant trait. Since a chick develops during embryonic life by utilizing egg yolk from the yolk sac, the variation of OTC activity among chicken breeds and within a breed in 2-day-old chicks seems to depend on a genetically controlled difference of inducibility by egg yolk. The Ocb is an autosomal gene which controls the induction of OTC activity, but it is difficult to explain the consistent difference in OTC activity between sexes by involving this gene or this locus alone.     

X-chromosome inactivation in human liver: confirmation of X-linkage of ornithine transcarbamylase.

Histochemical assay for ornithine transcarbamylase (OTC) activity in fixed frozen hepatic sections from a woman heterozygous for OTC deficiency revealed two populations of hepatocytes: those with normal activity and those with no activity. This observation, in conjunction with data from previous family studies, confirms the hypothesis that the gene for OTC is X-linked. It also provides the first cytologic demonstration of cellular mosaicism for a liver-specific cell product.     

Carrier detection in a partially dominant X-linked disease: ornithine transcarbamylase deficiency

Summary Ornithine transcarbamylase (OTC) deficiency is an X-linked disease responsible for lethal neonatal hyperammonemia in males. Partial OTC deficiency also occurs in females and can be responsible for life-threatening hyperammonemic comas in heterozygotes (15%). Increased orotic acid excretion occurs in both symptomatic and asymptomatic carriers, especially under protein loading tests. The disease is therefore partially dominant with neonatal lethality in the hemizygous male; the fraction of new mutations has previously been estimated to be low in males (point estimation = 0, upper bound of the confidence interval = 0.16) and 57% in females. Genetic counseling in this disease is difficult because it is not clear whether a negative protein loading test rules out carrier status. In an attempt to determine how reliable the test is for carrier detection, we investigated ten obligate carriers for orotic acid excretion; considering all data available, we concluded that the test is rarely negative in obligate carriers (8%). Consequently, a negative test in a mother decreases the minimum risk of being a carrier from 84% a priori to 30% if she had an affected son and from 43% a priori to 5% if she had a heterozygous daughter. Finally, the diagnosis of a new mutation in the germ cells of the maternal grandfather in one particular family could be ascertained by extensive DNA analysis.     

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 substitutions, 24 (38%) were G to A with the next most common being C to T (16%) and A to T (11%).