From the beginning of the 1990's, the analysis target for diseases associated with genetic factors has shifted from monogenic disorders and mutation to genetic variations associated with multifactorial susceptible genes. With the progressing analysis of personal genomes, it has been discovered that there are several million differences of the single nucleotide polymorphisms (SNPs) between individuals. These differences of nucleotides are proven to relate with the onset of the common disease. Although, genome-wide association study (GWAS) is mostly used to analyze common diseases with common variants, it is necessary to analyze the rare variants that contribute diseases with larger effect size to improve the effectiveness of medical science. For next-generation medicine and personalized medicine using genomic information, it is important to seek the association between diseases and endophenotypes. In this study, we analyzed the etiology of glaucoma from the genetic point of view.
To date, Glaucoma loci was identified through analyzing mainly primary open-angle glaucoma (POAG) and normal-tension glaucoma (NTG) families with a Mendelian inheritance pattern using traditional linkage analysis. In 2007, it was reported that, using GWAS, LOXL1 gene was associated with exfoliation glaucoma. The advent of GWAS and the rapidly growing catalogue of variants provided further evidence that CDKN2B-AS1 gene and other genes are associated with POAG and NTG. We tested SNPs from the region of chromosome 2 that includes the GLC1B glaucoma locus for association with POAG and NTG in a Japanese population. Our data suggested that, in the Japanese population, Hexokinase 2 (HK2) rs678350 was associated with POAG/NTG, and NCK2 rs2033008 was associated with NTG. Immunohistochemistry showed that both Hk2 and Nck2 are expressed in the retinal ganglion cell layer, and were related with glaucoma etiology.
Recently, genetic analysis sifted from linkage analysis with microsatellite markers to whole genome analysis with next-generation sequencer (NGS). Whole exome sequencing (exome analysis) is an efficient strategy to selectively sequence the all coding regions of approximately 200,000 exons. In developmental glaucoma, CYP1B1 gene is the only disease causing gene. To explore the new developmental glaucoma gene, we did the whole exome sequencing using appropriate glaucoma family. After the screening for the CYP1B1 gene, the CYP1B1 negative subjects were analyzed on NGS. The results revealed that there was heterogeneity of the developmental glaucoma genes, and there is the possibility that the de novo mutation would also be the disease causing.
Succeeding whole exome sequencing was for the POAG/NTG genes. We did whole exome sequencing on one POAG family and two NTG families. We extracted the candidate gene from several tens to one hundred after filtering the base change using 1000 Genomes and SNP database. There was no candidate gene overlapped on the three families. The candidate genes overlapped on two families were transcription factor related with differentiation of neural cells and actin-related gene. Thus, this whole exome sequencing could be a useful technology for seeking new glaucoma genes.
To apply the genomic data to genome medicine, the relationship between genetics, environment, and diseases needs to be explored. We created a big Biobank and began to seek the glaucoma causing genes and biomarkers from the specimen in the Biobank. In the fiscal year 2013, we will do 1000 whole genome sequencing (more than 30 depths), and build the Japanese genome reference panel including an allele frequency of less than 0.5%. This will make it possible to investigate the disease causing genes. In the analysis, we will extract the specific SNP and disease-related SNP, and make the optimized Japanese DNA array. Combined with genomic information, health records and medical records, it is expected that in the near future more comprehensive studies should greatly advance our understanding of the genetic basis of glaucoma and ultimately provide more effective glaucoma prevention, diagnosis and therapy.
Nippon Ganka Gakkai Zasshi (J Jpn Ophthalmol Soc) 118: 216-240, 2014.