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Leber's hereditary optic neuropathy(LHON) is a maternally inherited mitochondrial disease of an acute or subacute bilateral loss of central vision. G to A substitutions at nucleotide position 11778 in mitochondrial DNA(mt DNA) have been identified in approximately 40% to 90% of patients. In this study, regions containing mt DNA 11778 mutations were analyzed by polymerase chain reaction(PCR), non-RI single strand conformation polymorphisms(SSCP) and direct sequencing. In 26 visually affected patients, mt DNA 11778 mutations were detected in 9 patients (36.4%). In one pedigree of a LHON patient(L-6), four unaffected family members had heteroplasmy of the 11778 mutation using non-RI SSCP. Ratios of the heteroplasmy between wild type and mutant mt DNAs can be detected in non-RI SSCP and accurately quantified by video densitometric analyzer. Two types of novel polymorphisms, 11696 G to A and 11719 A to G, in the mt DNA region were also found in this non-RI SSCP analysis. Non-RI SSCP is an efficient and accurate method for diagnosis of mt DNA 11778 mutations and quantifying heteroplasmy in patients with LHON and pedigrees.

Apert syndrome, acrocephalosyndactyly Type I, is an autosomal dominant craniosynostosis comprising acrocephaly, facial dysmorphism and severe syndactyly of the hands and feet. Missense mutations at codons 252 and 253 at 5'-end on exon 7 of fibroblast growth factor receptor (FGFR) 2 have been identified in a large number of patients with Apert syndrome. In this study, nucleotide sequences on the intron 6 were determined by vector ligation-PCR and direct sequencing. Five DNA samples from sporadic Apert syndrome were examined by non-RI SSCP and direct sequencing using a primer pair of intron 6 and exon 7. All cases of the syndrome showed abnormal banding pattern in the SSCP and missense mutations from Ser to Trp at codon 252 of the FGFR2 gene. The non-RI SSCP and direct sequencing of the FGFR2 exon 7 from genomic DNAs may be a useful and rapid molecular means for clinical diagnosis of Apert syndrome.

Craniofacial syndromes, including Crouzon syndrome, Pfeiffer syndrome, Jackson-Weiss syndrome, Apert syndrome and achondroplasia, have been indicated that syndromes were associated with mutations of fibroblast growth factor receptor (FGFR) gene families. In this report, seven Japanese patients with craniofacial syndromes, three Crouzon syndromes and four achondroplasias, were analyzed on FGFR2 and FGFR3 genes by non RI-SSCP (single strand conformation polymorphisms) and direct sequencing. Missense mutations of the FGFR3 exon 10, at codon 380 in two sporadic cases and codon 375 in two familial cases, were detected in all cases of achondroplasia. Mutations of the FGFR2 were noted in Crouzon and Apert syndromes. One of three Crouzon syndromes has a missense mutation at codon 342 on exon 9. Highly frequent mutations were clustered within some localized regions of the FGFR genes in craniofacial syndromes. Alterations in these receptors due to missense mutations would thus appear closely involved in pathogenesis of craniofacial syndrome. The non RI-SSCP and direct sequencing of the FGFR genes, shown in this report, may be an appropriate approach for diagnosis of these syndromes with extensive clinical application.

A deficiency of methylmalonyl-CoA mutase (MCM) results in methylmalonic acidemia, which is inherited as an autosomal recessive disease and is characterized by accumulation of precursors and abnormal derivatives of methylmalonyl-CoA in body fluids. Abnormal splicing with 13 base pairs (bp) insertion at MCM exons 2 and 3 junction in MCM transcripts and a homozygous point mutation, g to a transition, on 5 bp downstream exon 2 were detected in a proband with methylmalonic acidemia. The parents in the kindred were heterozygous carriers of the g to a transition in MCM intron 2. Non-RI single strand conformation polymorphisms (SSCP) was conducted to devise for analysis of this MCM mutation. This non-RI SSCP is considered to be useful diagnostic means with high potential for extended clinical application.

Differential diagnosis of herpes virus species is often required in various clinical courses, such as transplantation and immunocompromised patients. Pairs of common primers which enabled sufficient amplifying and differential diagnosis of herpes virus species were designed and nested PCR using the common primer pairs was conducted to evaluate for its clinical application. The PCR was shown to provide amplification and differential diagnosis of HSV-1, HSV-2, VZV and CMV. The results of the PCR were consistent with those from fluorescent antibody of herpes virus culture and isolation. From clinical vesicular specimens, the PCR enabled the direct detection of HSV-1, HSV-2, and VZV. The PCR using the common primer pairs offers speed in the differential diagnosing of herpes virus.

Detection of Candida albicans by the polymerase chain reaction (PCR) was conducted. Oligonucleotide primers to amplify a 180 bp fragment on the beta-tubulin gene of C. albicans were synthesized. PCR was conducted using standard strains (14 species and 32 strains) and strains isolated from clinical samples (8 species and 12 strains) in Kitasato University Hospital. The PCR specifically amplified DNA from C. albicans of all standard strains and neither any other Candida, other fungal, bacterial nor human DNA. In three samples of clinically isolated C. albicans strains, the positive bands were also detected. This PCR procedure is more specific and rapid than the conventional culture method and is applicable to clinical diagnosis of the C. albicans infections.

Human cytomegalovirus (CMV) in this study, a polymerase chain reaction (PCR) assay was developed to detect human cytomegalovirus (CMV) from urine specimens taken from renal transplant patients and one congenital CMV infection. Twenty eight urine specimens of 20 renal transplant patients were analyzed by the PCR. CMV infection was detected in 11 specimens (8 recipients). The incidence (40.0%) of PCR positive in the renal transplant patients was highest among the other conventional methods including fluorescent antibody study, anti-IgM EIA and complement fixation test. In two cases, CMV infection could be identified by PCR before antibody development. The PCR procedure is more sensitive and rapid than conventional methods for the diagnosis of CMV. Rapid diagnosis of CMV infection using PCR may be useful in clinical diagnosis and have therapeutic value.

A cDNA complementary to the parathyroid hormone related protein (PTHrP), the humoral hypercalcemia factor in malignancy, was recently isolated and sequenced. PTHrP expression in human carcinomas was examined (determined) by Northern blot hybridization, Southern blot hybridization and radioimmunoassay (RIA). Expression of PTHrP mRNA was detected in three out of four lung squamous cell carcinomas, two out of ten breast carcinomas and the one adenosquamous carcinoma of the maxilla. No clinical hypercalcemia was found in these PTHrP mRNA positive carcinomas. No expression of PTHrP was detected in normal human or rat tissues. In Southern blot hybridization, no amplification of PTHrP gene was found in PTHrP positive cases. An insertion in one allele of promotor region of the gene was identified in one PTHrP positive lung squamous cell carcinoma. The serum level of PTHrP was examined using human PTHrP (1-34) RIA in lung carcinomas. We found no correlation between the level of PTHrP and clinical hypercalcemia or the histopathological diagnosis. We discuss some problems of the PTHrP assay as a novel tumor marker for malignancies. A new RIA assay study using recombinant human PTHrP expressed in Escherichia coli is also reported.