These results expand the spectrum of CLCN5 mutations associated with this renal disorder and provide insight into possible structure-function relationships. For example, both the missense mutations are located within a short putative loop between two transmembrane domains, and our results suggest that this region may have an important functional role in the regulation of channel activity.

Of the upregulated S100 genes, calgranulin members of S100 genes (S100A8, S100A9, and S100A12) were most highly expressed in the acute phase. Only one S100 gene, the S100A13 gene, exhibited a significantly decreased expression level in the acute phase.

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.

No abstract

The cytosolic 67-kD protein in phagocytes (p67-phox) and B lymphocytes is one of essential components of the superoxide-generating system in these cells, and its defect causes an autosomal recessive type of chronic granulomatous disease (CGD). We performed mutation analysis of p67-phox mRNA from a CGD patient who lacks the protein and found an in- frame deletion from nucleotide 694 to 879, which corresponds to the entire sequence of exons 8 and 9. This sequence encodes one of two Src homology 3 domains and a part of proline-rich domain in p67-phox and lack of these domains seem to have influenced stability of this protein. To know causative reason for the deletion, we analyzed genomic DNA for p67-phox using two sets of primers that covered exons 8 and 9 with adjacent introns. The DNA fragments from the patient were shown to be same in length as those from control. However, the single-strand conformation-polymorphism analysis of the fragments showed that a patient's specimen that included the splice junction of exon 9 exhibited different mobility from the control. By sequencing of the fragment, a homozygous G to A replacement at position +1 of intron 9 was found to be a sole mutation, which reduced the matching score of the splicing sequence to the consensus calculated according to the formula proposed by Shapiro and Senapathy (Nucleic Acids Res 15:7155, 1987). The reduced matching score at the splice doner site (5′ splice site) of intron 9 and the original low matching score at the acceptor site (3' splice site) of intron 7 may explain the skipping of exon 8 and 9, and another predicted mechanism is discussed on the basis of Shapiro and Senapathy's hypothesis.