Novel mutation in UMPS gene leads to false positive result of DUMPS (genetic disorder) in buffaloes: need for gene sequencing before confirming results of RFLP in new species.

Deficiency of uridine monophosphate synthase (DUMPS) is a lethal genetic disorder associated with early embryonic mortality. Murrah and Mehsana male buffaloes (n = 594) were screened for DUMPS by PCR-RFLP technique. A few Murrah buffalo male calves were found to be carriers of DUMPS in RFLP, which has not been reported earlier. On the Sanger sequencing, a novel A to G substitution mutation was identified in AvaI restriction recognition site of UMPS gene in buffaloes. This mutation hinders digestion of DNA by AvaI which leds to false positive results for DUMPS carrier in RFLP. The results indicated that genome sequencing must be performed before confirming results of RFLP in any new species. All the buffaloes that were tested had only wild-type genotype in exon 5 for DUMPS specific allele.

Conditional disruption of hepatic carbamoyl phosphate synthetase 1 in mice results in hyperammonemia without orotic aciduria and can be corrected by liver-directed gene therapy.

Carbamoyl phosphate synthetase 1 (CPS1) is a urea cycle enzyme that forms carbamoyl phosphate from bicarbonate, ammonia and ATP. Bi-allelic mutations of the CPS1 gene result in a urea cycle disorder presenting with hyperammonemia, often with reduced citrulline, and without orotic aciduria. CPS1 deficiency is particularly challenging to treat and lack of early recognition typically results in early neonatal death. Therapeutic interventions have limited efficacy and most patients develop long-term neurologic sequelae. Using transgenic techniques, we generated a conditional Cps1 knockout mouse. By loxP/Cre recombinase technology, deletion of the Cps1 locus was achieved in adult transgenic animals using a Cre recombinase-expressing adeno-associated viral vector. Within four weeks from vector injection, all animals developed hyperammonemia without orotic aciduria and died. Minimal CPS1 protein was detectable in livers. To investigate the efficacy of gene therapy for CPS deficiency following knock-down of hepatic endogenous CPS1 expression, we injected these mice with a helper-dependent adenoviral vector (HDAd) expressing the large murine CPS1 cDNA under control of the phosphoenolpyruvate carboxykinase promoter. Liver-directed HDAd-mediated gene therapy resulted in survival, normalization of plasma ammonia and glutamine, and 13% of normal Cps1 expression. A gender difference in survival suggests that female mice may require higher hepatic CPS1 expression. We conclude that this conditional murine model recapitulates the clinical and biochemical phenotype detected in human patients with CPS1 deficiency and will be useful to investigate ammonia-mediated neurotoxicity and for the development of cell- and gene-based therapeutic approaches.

De novo pyrimidine synthesis is necessary for intestinal colonization of Salmonella Typhimurium in chicks.

pyrE (STM3733) encodes orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10), the fifth enzyme of the de novo pyrimidine biosynthetic pathway. We identified a ΔpyrE mutant as under selection in screening of a Salmonella mutant library in 4-day old chicks. Here, we confirm that a ΔpyrE mutant colonizes 4-day old chicks poorly in competitive infection with isogenic wild type, and that the ability of this mutant to colonize chicks could be restored by providing a copy of pyrE in trans. We further show that our ΔpyrE mutant grows poorly in nutrient poor conditions in vitro, and that the ability of this mutant to grow is restored, both in vitro and in chicks, when precursors to the pyrimidine salvage pathway were provided. This finding suggests that the environment in the chick intestine during our infections lacks sufficient precursors of the pyrimidine salvage pathway to support Salmonella growth. Finally, we show that the colonization defect of a ΔpyrE mutant during infection occurs in to chicks, but not in CBA/J mice or ligated ileal loops in calves. Our data suggest that de novo pyrimidine synthesis is necessary for colonization of Salmonella Typhimurium in the chick, and that the salvage pathway is not used in this niche.

Mild orotic aciduria in UMPS heterozygotes: a metabolic finding without clinical consequences.

BACKGROUND: Elevated urinary excretion of orotic acid is associated with treatable disorders of the urea cycle and pyrimidine metabolism. Establishing the correct and timely diagnosis in a patient with orotic aciduria is key to effective treatment. Uridine monophosphate synthase is involved in de novo pyrimidine synthesis. Uridine monophosphate synthase deficiency (or hereditary orotic aciduria), due to biallelic mutations in UMPS, is a rare condition presenting with megaloblastic anemia in the first months of life. If not treated with the pyrimidine precursor uridine, neutropenia, failure to thrive, growth retardation, developmental delay, and intellectual disability may ensue. METHODS AND RESULTS: We identified mild and isolated orotic aciduria in 11 unrelated individuals with diverse clinical signs and symptoms, the most common denominator being intellectual disability/developmental delay. Of note, none had blood count abnormalities, relevant hyperammonemia or altered plasma amino acid profile. All individuals were found to have heterozygous alterations in UMPS. Four of these variants were predicted to be null alleles with complete loss of function. The remaining variants were missense changes and predicted to be damaging to the normal encoded protein. Interestingly, family screening revealed heterozygous UMPS variants in combination with mild orotic aciduria in 19 clinically asymptomatic family members. CONCLUSIONS: We therefore conclude that heterozygous UMPS-mutations can lead to mild and isolated orotic aciduria without clinical consequence. Partial UMPS-deficiency should be included in the differential diagnosis of mild orotic aciduria. The discovery of heterozygotes manifesting clinical symptoms such as hypotonia and developmental delay are likely due to ascertainment bias.

Hereditary Orotic Aciduria and the Excretion of Orotidine.

Objective Orotic aciduria and deficiency of uridine monophosphate synthetase have been observed in a patient, studied over 10 years, who had no megaloblastic anemia. Excretion of orotic acid and orotidine were 8.24 and 0.52 mmol/mol of creatinine. The ratio of 15.85 differed appreciably from that of 6 patients reported with no megaloblastic anemia. Methods The analysis of orotidine by gas chromotography mass spectrometry was conducted. Conclusion Patients with orotic aciduria with and without megaloblastic anemia cannot be distinguished by ratio of orotic acid to orotidine.

Hereditary orotic aciduria with epilepsy and without megaloblastic anemia.

Hereditary orotic aciduria is a rare metabolic disease that results from a defect of uridine-5-monophosphate synthase (UMPS). In affected patients, main clinical symptoms are a markedly increased urinary excretion of orotic acid combined with megaloblastic anemia. This report describes a new case of UMPS deficiency without megaloblastic anemia but with epilepsy.

Incidence of bovine leukocyte adhesion deficiency, complex vertebral malformation, and deficiency of uridine-5-monophosphate synthase carriers in Brazilian Girolando cattle.

Among the various hereditary diseases that have been widely studied in dairy cattle, bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine-5-monophosphate synthase (DUMPS), and complex vertebral malformation (CVM) are noteworthy because of their high impact on overall herd productivity as a consequence of increased calf mortality. The aim of this study was to verify the frequency of carriers of BLAD, CVM, and DUMPS mutant alleles in cows and bulls from the National Girolando Progeny Test carried out in Brazil by using polymerase chain reaction (PCR)-restriction fragment length polymorphism and allele-specific PCR assays. A total of 777 animals were genotyped for BLAD, 783 for CVM, and 122 for DUMPS. The frequencies of carriers for BLAD and CVM were 0.77 and 1.53%, respectively, whereas no carriers of DUMPS were observed.

Megaloblastic anemia--a rare cause.

A 2- year- old boy presented with non responsive megaloblastic anemia, growth failure and developmental delay. Blood levels of B(12), folic acid and iron were normal. Tandem mass spectroscopy for common inborn errors of metabolism did not reveal any abnormality. There was an increased excretion of orotic acid in urine. The authors report this as a rare cause of megaloblastic anemia.

Screening for bovine leukocyte adhesion deficiency, deficiency of uridine monophosphate synthase, complex vertebral malformation, bovine citrullinaemia, and factor XI deficiency in Holstein cows reared in Turkey.

BACKGROUND: Bovine leukocyte adhesion deficiency (BLAD), deficiency of uridine monophosphate synthase (DUMPS), complex vertebral malformation (CVM), bovine citrullinaemia (BC) and factor XI deficiency (FXID) are autosomal recessive hereditary disorders, which have had significant economic impact on dairy cattle breeding worldwide. In this study, 350 Holstein cows reared in Turkey were screened for BLAD, DUMPS, CVM, BC and FXID genotypes to obtain an indication on the importance of these defects in Turkish Holsteins. METHODS: Genomic DNA was obtained from blood and the amplicons of BLAD, DUMPS, CVM, BC and FXID were obtained by using PCR. PCR products were digested with TaqI, AvaI and AvaII restriction enzymes for BLAD, DUMPS, and BC, respectively. These digested products and PCR product of FXID were analyzed by agarose gel electrophoresis stained with ethidium bromide. CVM genotypes were detected by DNA sequencing. Additionally, all genotypes were confirmed by DNA sequencing to determine whether there was a mutant allele or not. RESULTS: Fourteen BLAD, twelve CVM and four FXID carriers were found among the 350 Holstein cows examined, while carriers of DUMPS and BC were not detected. The mutant allele frequencies were calculated as 0.02, 0.017, and 0.006 for BLAD, CVM and FXID, respectively with corresponding carrier prevalence of 4.0% (BLAD), 3.4% (CVM) and 1.2% (FXID). CONCLUSION: This study demonstrates that carriers of BLAD, CVM and FXID are present in the Turkish Holstein population, although at a low frequency. The actual number of clinical cases is unknown, but sporadic cases may appear. As artificial insemination is widely used in dairy cattle breeding, carriers of BLAD, CVM and FXID are likely present within the population of breeding sires. It is recommended to screen breeding sires for these defective genes in order to avoid an unwanted spread within the population.

Orotic aciduria and uridine monophosphate synthase: a reappraisal.

Three subtypes of hereditary orotic aciduria are described in the literature, all related to deficiencies in uridine monophosphate synthase, the multifunctional enzyme that contains both orotate: pyrophosphoryl transferase and orotidine monophosphate decarboxylase activities. The type of enzyme defect present in the subtypes has been re-examined by steady-state modelling of the relative outputs of the three enzymic products, uridine monophosphate, urinary orotic acid and urinary orotidine. It is shown that the ratio of urinary outputs of orotidine to orotate provides a means of testing for particular forms of enzyme defect. It is confirmed that the type I defect is caused by loss of uridine monophosphate synthase activity. Cells and tissue of type I cases have a residual amount of activity that is qualitatively unchanged: the relative rates of the transferase and decarboxylase do not differ from those of wild-type enzyme. The single claimed case of type II, thought to be due to specific inactivation of orotidine monophosphate decarboxylase, is shown to have a product spectrum inconsistent with that claim. It is proposed that this type II form does not differ sufficiently to be accepted as separate from type I. The third subtype, hereditary orotic aciduria without megaloblastic anaemia, occurs in two cases. It has the product spectrum expected of a defect in orotidine monophosphate decarboxylase. This form is the only one that appears to have a qualitatively different uridine monophosphate synthase. The possibility that orotidine monophosphate may control flux through the pyrimidine biosynthesis pathway in hereditary orotic aciduria is discussed.

Japanese black cattle with orotic aciduria detected by gas-chromatography/mass-spectrometry.

A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.

Lack of carriers of citrullinaemia and DUMPS in Indian Holstein cattle.

The present study investigated the occurrence of 2 autosomal recessive genetic diseases, bovine citrullinaemia and deficiency of uridine monophosphate synthase (DUMPS), in Indian Holstein cattle. The polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis was performed on a group of 642 animals, mainly HF and HF crossbred cattle, to identify carriers of these diseases. None of the animals were carriers of citrullinaemia or DUMPS. It is possible that with the mounting selection pressure, the international gene pool may diminish, and consequently the risk of dissemination of inherited defects will increase. It is therefore recommended to screen breeding bulls for their breed-specific genetic diseases before they are inducted in artificial insemination programmes, to minimize the risk.

Deficiency of uridine monophosphate synthase (DUMPS) and X-chromosome deletion in fetal mummification in cattle.

Ten mummified fetuses were tested for the deficiency of uridine monophosphate synthase (DUMPS), which is known to contribute to the embryonic and fetal mortality in cattle. Genomic DNAs of the mummified fetuses were extracted from tissue samples collected from the mummies and were amplified by GenomiPhi DNA amplification kit. UMPS gene of the mummies was amplified by polymerase chain reaction (PCR) with DUMPS primers. Out of ten mummies examined, two fetuses were heterozygous (carriers) for DUMPS as indicated by the presences of three bands of 89, 53 and 36 bp. Estimated stage of gestation when the death occurred in the two mummies was 3.5 and 2.5 months, respectively. The other fetuses exhibited only two bands of 53 and 36 bp on the polyacrylamide gel indicated that they were normal. On the other hand, all the mummies were sexed using AMX/Y primers. Specific regions of Y and X chromosomes were amplified by PCR using AMX/Y. The expected 280 bp fragment in the female sample and the 280 and 217 bp in the male sample were observed. Nine mummies had a normal X and Y chromosome bands; however, the other mummified fetus exhibited only Y chromosome band, while the constitutive X chromosome fragment was missing. The estimated stage of gestation when the death occurred in this mummified fetus was 100 days. This might be the first report of DUMPS and X-chromosome deletion at the amelogenin gene in bovine-mummified fetuses in Japan.

No incidence of DUMPS carriers in Polish dairy cattle.

DUMPS (Deficiency of Uridine Monophosphate Synthase) is a hereditary recessive disorder in Holstein cattle causing early embryo mortality during its implantation in the uterus. The only way to avoid the economic losses is early detection of DUMPS carriers. Because American Holstein semen has been intensively imported to Poland since 1970, there was a risk that DUMPS could have spread in Polish dairy cattle. In our study, 2209 dairy cattle of the Polish Holstein breed have been screened by the DNA test. The dominant group was young bulls entering the testing program (1171) and proven bulls (781). They represented all sires entering Polish breeding programs between 1999 and 2003. Also, 257 sire dams were included in the screening program. No DUMPS carrier has been found. Our results then indicate that the population of dairy cattle reared in Poland is free from DUMPS. Because of the economical significance of the DUMPS mutation and its recessive mode of inheritance, attention has to be paid to any case of a bull having in his origin any known DUMPS carrier. Such a bull should be tested and if positive eliminated from the active population. Also, young bulls (testing bulls) should be screened for DUMPS if in their progeny a high incidence of embryo mortality is observed and their genealogy cannot exclude their relatedness to any DUMPS carriers.

Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, Cyanidioschyzon merolae 10D.

Although the nuclear genome sequence of Cyanidioschyzon merolae 10D, a unicellular red alga, was recently determined, DNA transformation technology that is important as a model plant system has never been available thus far. In this study, improved culture conditions resulted in a faster growth rate of C. merolae in liquid medium (doubling time = 9.2 h), and colony formation on gellan gum plates. Using these conditions, spontaneous mutants (5-fluoroortic acid resistant) deficient in the UMP synthase gene were isolated. The lesions were then restored by introducing the wild-type UMP synthase gene into the cells suggesting DNA transformation by homologous recombination.

Decreased fluorouracil cytotoxic effect on EB-virus transformed lymphocytes from hereditary orotic aciduria.

5-Fluorouracil (5-FU), used widely for malignancies, phosphorylate mostly by uracil phosphoribosyl transferase (UPRT). Patients with hereditary orotic aciduria lack the orotate phosphoribosyl transferase (OPRT) activity. In the cancer cells, the OPRT activity is paralleled with the UPRT activity. This study shows that the UPRT activity of the hereditary orotic aciduria homozygote decreased about 40% of normal controls. Moreover, we investigated the 5-FU cytotoxic effects on hereditary orotic aciduria (one homozygote, 4 heterozygotes and 7 normal controls), using EB-virus transformed lymphocytes (EB-LC). 5-FU was addded to the culture medium at concentrations ranging from 0 to 10.0 micromol/l. The 5-FU cytotoxic effects on the homozygote were milder than those on controls at each 5-FU concentration. The 5-FU cytotoxic effects in the heterozygotes were at intermediate levels between the homozygote and controls. We speculate that 5-FU cytotoxic effects, both anti-tumor effects and adverse reactions, would be weak when a patient with hereditary orotic aciduria was treated with 5-FU.