PubMed2004
Human diseases develop by complex mutual relationships of genetic and environmental factors. In inherited diseases, DNA diagnosis of the disease-causing genes provides a confirmation of the disease. On the other hand, DNA diagnosis of the disease-sensitive genes in multifactorial diseases, such as the lifestyle-related diseases (common diseases), provides the risk of developing the disease. Two new technologies are being used for DNA diagnosis in the clinic. The first is called Invader Technology and is a non-PCR method and is useful for detecting well-known genetic variations in large samples efficiently. We have developed a method to quantify the heteroplasmy of mitochondrial DNA mutations by this technique. The second technique, called WAVE, uses denaturing high-performance liquid chromatography to screen for mutations in a large number of samples automatically and efficiently. Clinical DNA diagnoses are divided into those for single genetic diseases and those for multifactorial diseases. The purposes for DNA diagnosis in single genetic diseases are: 1) to propose a new clinical classification of the disease, such as TGFB1-related corneal dystrophy or retinitis pigmentosa, based on the genotypes; 2) to confirm a clinical diagnosis, such as Leber's hereditary optic neuropathy (LHON); and 3) to provide an early diagnosis before the development of the disease and thus provide an opportunity to start early treatment. For example, a family history of glaucoma is one of the risk factors for developing glaucoma. The frequency of mutations in the glaucoma genes, myocilin and optineurin, were found to be about 3% and 0.25%, respectively, in Japanese. The significance of DNA diagnosis in multifactorial diseases is that it provides a risk diagnosis for an individual. Single nucleotide polymorphisms (SNPs) of disease-sensitive genes are associated with only a 2- to 3-fold risk of developing the disease. A case-control association study was performed using many SNP markers to identify glaucoma-sensitive genes. A total of 671 Japanese individuals, 201 POAG patients, 234 NTG patients, and 236 normal controls were examined. Fifty-two SNPs in the 38 genes were examined to identify the glaucoma-sensitive genes as candidate genes, and SNPs in AT 1, AT 2, PON 1, GSTT 1, NOS 3, and EDN 1 were associated with glaucoma statistically. Mitochondrial (mt) DNA mutations associated with LHON might be risk factors for open-angle glaucoma, because abnormal optic disc excavations are also found in LHON patients. A total of 651 blood samples were screened for 6 LHON-associated mutations with the Invader assay. Seven patients had one of the five mutations, but none had developed LHON. The 5 mutations were not identified in 236 normal controls. MtDNA mutations may make the optic disc more susceptible to damage in glaucoma patients. The clinical variability in LHON patients suggests that the disease most likely results from multi factorial mechanisms. To determine whether genetic polymorphisms for oxidative stress and apoptosis cause clinical variability in patients with LHON, 12 polymorphisms in 10 genes were analyzed in 87 patients with the 11778 mutation in relation to the age at onset and final visual acuity. LHON patients carrying homozygous His 113 in the EPHX1 gene or homozygous Arg 72 in the TP53 gene developed the disease earlier than those without this genotype. Thus, nuclear genetic polymorphisms related to oxidative stress or apoptosis may modify the age of LHON onset. A clinical trial of 38 healthy volunteers without systemic diseases or eye diseases was performed using an angiotensin II receptor blocker (candesartan cilexetil) as an alternative drug for lowering intraocular pressure (IOP). After a single oral dose of candesartan cilexetil, the IOP fell significantly for 24 hr. There was no association between the effects of oral candesartan cilexetil and the three SNPs in the AT 1 gene. In the 21th century, DNA diagnosis for multifactorial diseases will be required to determine the treatment plan for individuals or to prevent diseases. We have developed a panel of tests by Invader assay for clinical use to detect mutations in the myocilin gene or in LHON. In the future, we will develop a panel to detect SNPs in the glaucoma-sensitive genes to diagnose individuals at risk for developing glaucoma. Such information is expected to help develop new medications.