Prenatal diagnosis of ornithine transcarbamylase deficiency utilizing fetal liver biopsy.

The prenatal diagnosis of ornithine transcarbamylase deficiency (OTCD) was made by using radioactive microassays for ornithine transcarbamylase (OTC) and--as an internal control--carbamyl phosphate synthetase (CPS I) in liver biopsy material from two 19-week-old at-risk fetuses. In each case, no OTC activity could be detected, while CPS I activity was normal. Control fetuses of 17-21 weeks gestation had OTC activities in the range of 10.7 to 19.4 mumol/mg protein per hr. The prenatal results were confirmed post abortum by the radiochemical assays and by an enzyme-specific cytochemical staining method.     

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.     

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.     

Molecular detection and correction of ornithine transcarbamylase deficiency

The application of new diagnostic techniques has led to improvement in carrier detection and prenatal diagnosis in ornithine transcarbamylase deficiency. Progress has also been made towards somatic gene therapy.     

Prenatal diagnosis using fetal cells from the maternal circulation.

All current methods of fetal karyotyping are invasive and carry a definite, albeit small, procedure-related risk. Because of this and testing costs, only women older than 35 years who have a greater risk for fetal aneuploidy are currently offered prenatal testing. But this detects only 20% to 25% of fetuses with Down syndrome. It would be a tremendous advance to find a noninvasive technique for prenatal diagnosis that carries no procedure-related risk and could be offered to all pregnant women. We describe a possible technique for noninvasive prenatal diagnosis that aims to identify fetal cells in the peripheral maternal circulation and successfully garner them for prenatal testing. Early attempts at fetal karyotyping were hampered by inaccurate diagnostic methods and cumbersome cell-counting techniques. Today, improved capabilities of identifying and enriching for fetal cells, coupled with sensitive methods of analysis such as the polymerase chain reaction, bring renewed enthusiasm to this task. Many technical issues, as well as serious questions regarding the test''s utility, still exist, however, and must be explored and answered before the capture of fetal cells in the maternal circulation translates into reality for noninvasive prenatal diagnosis.     

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.     

Correction of mouse ornithine transcarbamylase deficiency by gene transfer into the germ line

The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.     

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%).     

A novel missense mutation in exon 8 of the ornithine transcarbamylase gene in two unrelated male patients with mild ornithine transcarbamylase deficiency

We studied two unrelated male probands with mild ornithine transcarbamylase (OTC) (E.C.2.1.3.3) deficiency presenting a similar clinical course. Previous analyses of their liver OTCs also revealed similar properties. To identify the underlying molecular defects, we first cloned the entire coding region of the OTC gene from one proband and found a single base-substitution (C to T) leading to the substitution of tryptophan for arginine at amino acid position 277. Using a genomic amplification technique followed by allele specific oligonucleotide hybridization, we identified the same point mutation in the OTC gene of the other proband. We observed the presence of the mutation among family members in at least three generations, and in one asymptomatic hemizygous sibling in each family.     

Prenatal sex determination from maternal peripheral blood using the polymerase chain reaction

We have investigated the use of a nested polymerase chain reaction assay for the detection of a fetal-specific Y-chromosomal sequence (DYS14) from DNA extracted from unsorted maternal peripheral blood. Serial dilutions of male DNA into female cord blood DNA indicated that the assay could detect an equivalent of a single male cell in 300000 female cells. The assay exhibited absolute specificity for male DNA with no amplification from a DNA panel obtained from 10 female cord blood samples. When used on DNA extracted from unsorted peripheral blood from a series of pregnant women, the predictive values of a positive test for a male fetus were 86%, 67% and 87% in the first, second and third trimesters, respectively. We have also demonstrated that retesting the samples allows the detection of a proportion of male-bearing pregnancies with a high degree of accuracy, in that all 15 women who gave positive signals in two consecutive amplifications had male fetuses. We have also applied the test at 8 weeks post-partum to eight women who had previously delivered male babies; no Y-specific signal could be detected in any of them, suggesting that most women have cleared their circulation of fetal cells by 8 weeks after parturition.     

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.     

Enrichment of fetal trophoblasts and nucleated erythrocytes from maternal blood by an immunomagnetic colloid system

The aim of this study was to isolate fetal trophoblasts and nucleated erythrocytes from maternal blood using the immunomagnetic colloid system. About 25 ml of maternal blood was collected from pregnant women between of 14 and 20 weeks gestation. Nucleated erythrocytes (NRBCs) were isolated from 5 ml of maternal blood and a nested polymerase chain reaction for the Y chromosome was used to determine fetal origin. The sensitivity of the fetal gender diagnosis was 80% and the specificity was 86%. Both fetal trophoblasts and NRBCs were isolated from the remaining 20 ml of maternal blood. The fetal gender of the trophoblast-enriched fraction was determined using fluorescence in situ hybridisation (FISH) with dual-colour XY-specific DNA probes. XY-specific signals were observed in 0.38% of cells sorted from all pregnant women carrying male fetuses (n = 10). Simultaneous immunophenotyping for the fetal haemoglobin and FISH using XY probes were used to evaluate the fetal origin of cells enriched with anti-CD71. The mean percentage of male fetal erythroblasts was 0.24% and the number of fetal erythroblasts was estimated to be about 672 in 20 ml of maternal blood. The number of fetal erythroblasts detected in our study was greater than that detected by most other separation techniques. Our study shows that it would be feasible to use the immunomagnetic colloid system for the isolation of both trophoblasts and NRBCs from the same maternal blood sample with relatively good efficiency. Received: 17 December 1998 / Accepted: 9 February 1999     

Fetal gender determination and BclI polymorphism using nucleated erythrocytes in maternal blood.

This study demonstrated determination of fetal gender from nucleated red blood cells (NRBCs) in maternal blood and attempted to apply prenatal diagnosis of hemophilia A using BclI DNA polymorphism. Venous blood was drawn from 20 pregnant women, and NRBCs were recovered by magnetic activated cell sorting and anti-GPA (glycophorin A) immunostaining. After microdissector isolation of the NRBCs, primer extension preamplification (PEP) and nested PCR of the amelogenin gene were performed to determine fetal gender. We also performed PEP and nested PCR of BclI polymorphism to verify the validity of prenatal diagnosis of hemophilia A. DNA amplification was achieved in 107 cells (51.9%) and fetal gender determined with 65.0% accuracy. Unfortunately, we could not verify the validity within the scope of this study. However, in a larger number of cases that are informative in BclI polymorphism, we will be able to identify patients affected by hemophilia A using fetal NRBCs in maternal blood.     

母体外周血中胎儿有核红细胞的分离和富集方法的研究进展

母体外周血中分离的胎儿有核红细胞（fNRBCs）包含胎儿完整的遗传信息,可用于无创产前诊断。fNRBCs的分离和富集方法主要分为三类：物理分选法、抗原-抗体结合分离法和增殖法。不同的方法获得的fNRBCs的数量和纯度不同,多种方法联合使用可以提高富集产物中fNRBCs的纯度和数量。本文就母体外周血中fNRBCs的分离和富集方法进行综述。     

Correction of ornithine transcarbamylase (OTC) deficiency in spf-ash mice by introduction of rat OTC gene

We introduced rat ornithine transcarbamylase (OTC) gene into OTC-deficient spf-ash mice by mating spf-ash heterozygotes with transgenic mice which carried recombinant DNA composed of 1.3 kb of the 5' flanking region of the gene fused onto rat OTC cDNA. The liver OTC activity of hemizygous spf-ash mice which carried the transgene was about twice that of nontransgenic spf-ash mice, and the small intestinal OTC activity was 6 times higher; the values being 12% and 27% of the control levels, respectively. The transgenic spf-ash mice showed normal hair growth without sparse fur, nearly normalized urinary orotic acid excretion and normalized serum citrulline concentration.