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

Abstract

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.

Fetal cells in the maternal circulation: feasibility for prenatal diagnosis

“…Micromanipulation of individual or pooled NRBCs, followed by PCR, has been advocated as a means of fetal genotyping for conditions in which the mother carries a mutant allele. This technique was first described by Takabayashi et al (1995) but was applied to the diagnosis of Duchenne muscular dystrophy, Rhesus D (RhD), HLA‐DQ alpha genotype, and ornithine transcarbamylase deficiency by Sekizawa et al (1996a, b, 1998) and Watanabe et al (1998). Cheung et al (1996) identified fetal cells by a combination of MACS and anti‐gamma or anti‐zeta staining, and pooled several NRBCs to prevent the complication of allele dropout.…”

Optimization of nucleated red blood cell (NRBC) recovery from maternal blood collected using both layers of a double density gradient

“…For this reason, most laboratories are currently combining one or more strategies to separate fetal cells from maternal blood. Density gradient centrifugation (Oosterwijk et al, 1996;Sekizawa et al, 1999a), selective cell lysis (Saunders et al, 1997), micromanipulation of individual cells (Sekizawa et al, 1996;Watanabe et al, 1998;Sekizawa et al, 1999b),¯ow sorting (Herzenberg et al, 1979;Lewis et al, 1996;Sohda et al, 1997), magnetic activated cell sorting (MACS) (Gaenshirt-Ahlert et al, 1992;Bu È sch et al, 1994), charge¯ow separation (Wachtel et al, 1996) and various combinations thereof have been used to enrich target cells by reducing the ratio of maternal cells to NRBC (Oosterwijk et al, 1998a). Simple, double or triple density gradients are widely employed as a common ®rst step or a unique step in the enrichment of NRBC from maternal blood.…”

Enrichment and detection of fetal erythroid cells from maternal peripheral blood using liquid culture

“…Such techniques have included uorescence-activated cell sorting (FACS), MACS, antibody-conjugated columns, and micromanipulation of individual cells (Lewis et al, 1996;Watanabe et al, 1998;de Graaf et al, 1999). Most investigators begin with y20 ml maternal peripheral blood and after density gradient centrifugation, various positive and negative selection techniques are performed using speci®c monoclonal antibodies.…”