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Discovery of a Strong Association Between common Sequence Variants in the LOXL1 Gene and Exfoliation Glaucoma in Iceland

Fridbert Jonasson, winner of WGA-Award 2007 'The Present' (together with Kari Stefansson)

Like other genome-wide association studies, this study has two main components. Firstly, there is a clinical component called phenotyping, a term sometimes used to describe a detectable effect of a gene, that in this case is a diagnosis. Secondly, there is a genetic component that includes genotyping and mathematical analysis as well as replication in an independent population to decrease the risk of false positive results. The genotypes found in clinical cohorts with different diagnosis are then compared with controls, much like in a case control study. In our study, the selection and phenotypic assessment of the population derived from the Reykjavik Eye Study a random sample from the national population census, was first performed in 1996 and repeated in 2001. The phenotyping created cohorts with exfoliation glaucoma (XFG), exfoliation syndrome (XFS) without glaucoma and primary open-angle glaucoma (POAG) as well as controls. Additionally, we recruited some of my surgical and clinical cases. For stability of diagnosis and contrary to common clinical practice we only included those with definite exfoliative material on the anterior lens capsule and had a separate cohort including those suspected of having exfoliative changes. The level of stability of diagnosis is important for genome-wide association studies and may be problematic in late onset diseases like XFS. Misclassification of participants can markedly reduce study power and bias study results towards no association particularly if unaffected patients are classified as affected.

The genetic part was done by the scientists of deCODE Genetics. The genome-wide association approach used to detect the lysyl-oxidase-like-1 (LOXL1) single nucleotide polymorphisms (SNPs) associated with XFS is revolutionary, because it permits interrogation of the entire human genome at levels of resolution previously unattainable. A SNP is a small genetic change or variation that can occur within a person's DNA sequence or nucleotide letter in the genetic code. It may be replaced by another letter which again may contribute to disease. In 2006 deCODE Genetics began examining concurrently several diseases including eye diseases using high density arrays containing more than 100,000 SNP's associated with disease. In 2007, when 195 cases of open-angle glaucoma had been genotyped a strong genome-wide significance was achieved (over-representation of sequence variants), in this 195 cases as compared to 14,474 controls using the Illumina 300 Hap chip and monitoring results. This Illumina chip includes probes for 317,000 SNPs and these SNPs tag as highly correlated surrogates about 80% of the genetic variance in persons of European descent. The genome-wide significance achieved was located on chromosome 15q24.1 and associated with an amino-acid switch in the protein. At this stage we were examining all open-angle glaucoma cases as one cohort. Further subtypes were, however, available from the Reykjavik Eye Study and when examined separately, it became evident that all the risk was associated with the exfoliation syndrome without glaucoma and exfoliation glaucoma, and not with primary open-angle glaucoma. All the risk was associated with two non-synonymous variants in the first exon of the LOXL1 gene, variants that are common in the Icelandic population, with 85% and 67% population allelic frequency, respectively. The protective version of the variants were not observed together on the same chromosome, suggesting that the risk variants could be ancestral.

Pair-wise comparison of the risk of the three observed haplotypes suggested that the persons with the high-risk variants were at 700 times higher risk of exfoliation syndrome than those with the low-risk haplotype, and combined the variants could explain more than 99% of the population attributable risk. The lower-risk exonic SNP variant was found to be associated with decreased expression of LOXL1 in adipose tissue in our study and this has since been confirmed in ocular tissue. The higher-risk exonic variant did, however, not affect expression. We did not find the LOXL1 gene polymorphisms to be associated with exfoliation syndrome suspects. Including XFS suspects in the XFS cohort would have markedly reduced the study power and biased the study results towards no association classifying large numbers of unaffected individuals as affected. Exfoliation syndrome, a disease of the extracellular matrix is con-sidered to be the most common identifiable cause of open-angle glaucoma. Elastosis of ocular tissues including the lamina cribrosa and outflow channels has been suggested to be a part of exfoliation glaucoma.

Our study is a strong support for a role of elastogenesis and elastosis in exfoliation syndrome demonstrating a highly significant association between DNA-sequence variants in the gene coding for LOXL1, a protein responsible for elastin synthesis and elastin fibre maintenance.

Our findings are the first big step towards understanding the genetic basis of open-angle glaucoma. This study shows how combining epidemiology and genetics, in particular genome-wide association studies, can help us solve difficult problems.