An Investigation Into LOXL1 Variants in Black South African Individuals with Exfoliation Syndrome

OPHTHALMIC MOLECULAR GENETICS

SECTION EDITOR: JANEY L. WIGGS, MD, PhD An Investigation Into LOXL1 Variants in Black South African Individuals With Exfoliation Syndrome

Robyn M. Rautenbach, MBChB, DipOphth(SA); Soraya Bardien, PhD; Justin Harvey, MCom(Mathematical Statistics); Ari Ziskind, MBChB, FCSOphth(SA), MSc, BSc

Objective: To investigate the association between 2 ly- ing majority of cases with XFS (PϽ.00001; odds ra- syl oxidase–like 1 (LOXL1) polymorphisms, rs1048661 tio,17.10; 95% confidence interval, 4.91-59.56), con- (R141L) and rs3825942 (G153D), and exfoliation syn- trary to all previous articles in which the GG genotype was drome (XFS) in black South African individuals. strongly associated with the disease phenotype.

Methods: A total of 43 black patients with XFS and 47 Conclusion: The LOXL1 SNPs R141L and G153D are ethnically matched controls were recruited for genetic significantly associated with XFS in this black South Afri- analysis. Samples were analyzed for presence of the can population. The AA genotype of G153D confers XFS LOXL1–R141L and G153D variants using restriction frag- risk in this population, as opposed to the GG genotype ment length polymorphism analysis. A case-control as- described in all other populations, suggesting that un- sociation study was performed. identified genetic or environmental factors indepen- dent of these LOXL1 SNPs may influence phenotypic ex- Results: The R141L and G153D single-nucleotide poly- pression of the syndrome. morphisms (SNPs) were both significantly associated with XFS (P=.00582 and PϽ.00001, respectively). Consis- Clinical Relevance: Elucidation of the role of genetic tent with findings in white populations but not in Asian factors, including the LOXL1 gene, in XFS will facilitate cohorts, the GG genotype of the R141L SNP was present identification of individuals predisposed to developing in significantly more XFS cases than controls (P=.00582). this condition. However, in this black South African study population, the AA genotype of G153D was present in an overwhelm- Arch Ophthalmol. 2011;129(2):206-210

XFOLIATION SYNDROME angle glaucoma, accounting for approxi- (XFS) is a generalized dis- mately 25% of cases worldwide.5 order of the extracellular The prevalence of XFS increases with matrix characterized by the age, and a number of studies have re- pathological deposition and ported geographical clustering of this con- accumulationE of fibrillar material through- dition based on race and ethnicity.6 Fa- out the eye. The origin of this fibrillar ma- milial aggregation studies have suggested terial is unknown but believed to be de- a significant genetic contribution to XFS.7 rived from abnormal basement membranes Despite these findings, a simple inherit- Author Affiliations: Division of of aging epithelial cells in ocular struc- ance model is not evident, suggesting that 1 Ophthalmology, Stellenbosch tures. In addition to its occurrence within XFS is the result of a complex inherit- University and Tygerberg the eye, exfoliative fibrillopathy has been ance pattern with multiple contributing ge- Hospital (Drs Rautenbach and reported in the skin, blood vessels, and vis- netic and/or environmental factors. Ziskind); and Division of ceral organs, suggesting that XFS may in A landmark genome-wide association Molecular Biology and Human fact be an ocular manifestation of a sys- study by Thorleifsson et al8 identified 3 Genetics, Faculty of Health temic disorder.2-4 common single-nucleotide polymor- Sciences (Dr Bardien) and This condition is associated with an ar- phisms (SNPs) in the lysyl oxidase–like Centre for Statistical Consultation, Department of ray of ocular manifestations, most fre- 1(LOXL1) gene on chromosome 15q24.1 Statistics and Actuarial Science, quently a severe and progressive form of that were strongly associated with XFS and Stellenbosch University chronic open-angle glaucoma. Exfolia- exfoliation glaucoma in Scandinavian (Dr Harvey), Cape Town, tion syndrome is acknowledged as the populations. The LOXL family of pro- South Africa. most common identifiable cause of open- teins play a vital role in the homeostasis

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 of elastic tissues, acting as cross-linking enzymes and to detect a clinically significant association between the pres- thereby ensuring spatially defined deposition of elastin ence or absence of a specific polymorphism and XFS. Data was fibrils.9,10 The identification of the LOXL1 protein in pseu- analyzed using SAS version 9.1 (SAS Institute Inc, Cary, North doexfoliation deposits verifies its involvement in abnor- Carolina). Descriptive statistics were computed for patient age, mal fibrinogenesis in pseudoexfoliative tissues.11,12 and comparison between the mean ages of cases and controls was performed by t test for 2 groups. Furthermore, the pri- Two of these SNPs, rs1048661 (R141L) and rs3825942 mary outcome variables were analyzed by contingency tables. (G153D), are located within exon 1 of LOXL1 and cause Associations between the cases and controls and the different amino acid missense changes in the protein. This exon allele, genotype, and haplotype frequencies was examined using codes for the N-terminal portion of the protein, which Pearson ␹2 test. Odds ratios and relative risk estimates were also may have a role in directing the LOXL1 protein to sites produced to examine further interactions between the disease of elastogenesis. The third LOXL1 SNP, rs2165241, is lo- and specific alleles and genotypes. Confidence intervals (CIs) cated in the first intron of the gene and is presumed not for these estimates were also produced. A PϽ.05 represented to have any biological consequence. All 3 of these SNPs statistical significance in hypothesis testing and 95% confi- were in significant linkage disequilibrium in the studied dence intervals were used to describe the estimation of un- population.8 These genetic findings have been repli- known parameters. Hardy-Weinberg equilibrium of the allele and genotype fre- cated to a large extent, with some important variations, 13-17 quencies of cases and control subjects was examined, both sepa- in numerous studies throughout North America, Aus- rately and in combination, using ␹2 and Fisher exact tests. tralia,18 Europe,19 and Asia.20-26 This study investigates the association of these LOXL1 gene polymorphisms with XFS among black South Af- RESULTS ricans, a geographical cluster with a high prevalence of XFS and exfoliation glaucoma.27,28 Of the 43 patients with XFS, 15 had exfoliation glaucoma. Of the 47 controls; 13 had primary open-angle glau- METHODS coma but no evidence of exfoliation. The cases and controls were age-matched with a mean (SD) age of 72.37 (9.57) in XFS cases and 71.81(7.56) in controls without PATIENT POPULATION XFS, with no significant difference found between the means of these 2 groups (P=.75621). An ethnically matched cohort of 43 elderly black patients with exfoliation syndrome and 47 control individuals were identi- The G allele of SNP rs1048661 (R141L) was detected fied from the outpatient ophthalmology service at the East Lon- in a statistically higher frequency in patients with XFS don Hospital Complex (Eastern Cape, South Africa) for this than controls (P=.00106). The relative risk of having no study. The study was approved by the Stellenbosch University disease given the presence of the G allele vs the T allele Committee for Human Research (N08/08/208), and all pa- for this SNP was 0.49 (95% CI. 0.42-0.57). The A allele tients and controls were recruited after informed consent. All of SNP rs3825942 (G153D) was strongly associated with cases and controls underwent an anterior segment evaluation exfoliation syndrome (PϽ.00001) in this sample, with after pupillary dilatation to confirm the presence or absence of patients being 9.94 times more likely to have an A allele the characteristic fibrillar material diagnostic of XFS. Venous than a G allele (odds ratio,9.94; 95% CI, 4.75-20.79) blood samples were collected from all study participants. (Table 1). The genotype frequencies for the rs1048661 (R141L) GENOTYPING SNP and the rs3825942 (G153D) SNP confirmed statistically significant differences between the XFS cases and DNA was extracted from the peripheral venous blood samples controls. The GG genotype of R141L was present in sig- according to established methods. Polymerase chain reaction primers were designed to amplify the region containing the nificantly more XFS cases than controls (P=.00582), with LOXL1 R141L and G153D variants (forward: 5Ј-GCA GGT GTA a relative risk of having no disease (GG vs GT/TT) CAG CTT GCT CA-3Ј and reverse: 5Ј-GGC CGG TAG TAC of0.46 (95% CI, 0.37-0.59). In this study population, we ACG AAA CC-3Ј), which produced a product of 474 base pairs. found that the AA genotype of G153D was present in an Restriction fragment–length polymorphism analysis was used overwhelming majority of cases with XFS (PϽ.00001), to genotype the 2 SNPs. The Smal (fermentas) and Eco24I (fer- with an odds ratio (AA vs GG) of 17.10 (95% CI, 4.91- mentas) restriction endonuclease enzymes were used for R141L 59.56) (Table 1). and G153D, respectively. Following digestion, the polymer- The haplotypes composed of the 2 LOXL1 SNPs ase chain reaction products were resolved on 12% polyacryl- rs1048661 and rs3825942 were determined, with the fre- amide gels and the bands visualized using silver staining. The quencies of the 2-SNP haplotypes differing significantly genotyping method was verified by sequencing randomly se- between the patients with XFS and the controls lected samples using the BigDye Terminator Sequence Ready Ͻ Reaction kit version 3.1 (Applied Biosystems, Foster City, Cali- (P .00001). The GA haplotype was associated with the fornia) and analyzed on a 3130xl Genetic Analyzer (Applied highest risk of XFS in which a patient is 9.94 times more Biosystems). BioEdit version 7.0.1 software was used for analy- likely to have XFS if haplotype GA is present than if either sis of the sequencing electropherograms.29 haplotypes TG or GG are present (odds ratio,9.94; 95% CI, 4.75-20.79). The TG haplotype was not detected STATISTICAL ANALYSIS among the group with XFS (Table 2). Hardy-Weinberg analysis of allele and genotype fre- A power analysis was performed prior to analysis of the data. quencies in cases and controls of both SNPs found Hardy- It was found that the sample size had sufficient power (85.345%) Weinberg equilibrium in all of the subpopulations, with

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©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 Table 1. LOXL1 Allele and Genotype Frequencies by Single-Nucleotide Polymorphism in XFS Cases and Controls

No. (%)

XFS Controls P Value (␹2) RR (95% CI) R141L (rs1048661) Allele G 86 (100) 83 (88.3) .00106 0.49 (0.42-0.57) T 0 (0) 11 (11.7) Genotype GG 43 (100) 37 (78.7) .00582 0.46 (0.37-0.59) GT 0 (0) 9 (19.1) TT 0 (0) 1 (2.1) G153D (rs3825942) Allele OR (95% CI) A 74 (86.0) 36 (38.3) .00001 9.94 (4.75-20.79) G 12 (14) 58 (61.7) Genotype AA 36 (83.7) 8 (17.0) .00001 17.10 (4.91-59.56) GA 2 (4.7) 20 (42.6) GG 5 (11.6) 19 (40.4)

Abbreviations: CI, confidence interval; OR, odds ratio; RR, relative risk; XFS, exfoliation syndrome.

A recent publication corroborates the findings of Table 2. LOXL1 Haplotypes in XFS Cases and Controls this study. Williams and colleagues31 investigated LOXL1 SNPs in black South African patients with rs1048661-rs3825942 exfoliation glaucoma and also found that the A (and Haplotype XFS Controls P Value (␹2) not the G allele) of G153D, in contrast to all other GA 74 36 Ͻ.00001 studies, is significantly associated with XFS and exfo- GG 12 47 TG 0 11 liation glaucoma in black South African individuals. An advantage of our compared with that of Williams Abbreviation: XFS, exfoliation syndrome. et al is that our study focused on only 1 ethnic sub- group, the Xhosa-speaking Bantu population (our cases and controls were recruited exclusively from an the exception of the G153D cases (P=.00002). This was ␹2 area where Xhosa-speaking black South African indi- confirmed using both and Fisher exact tests and after viduals predominantly reside). checking for genotyping errors. The allele frequencies observed in our study compare favorably with the data observed in HapMap (http://hapmap COMMENT .ncbi.nlm.nih.gov/) and dbSNP (http://www.ncbi.nlm.nih .gov/SNP/) as well as the aforementioned study of an- This study’s participants are representative of the South other black South African population.30 For G153D African Xhosa population, who represent the southern- (rs3825942), the G allele has been found at frequencies most extension of the Bantu-speaking nations that be- ranging from 58% to 63% in various black populations in gan to migrate southwards from an area near present- the Southwestern United States, Kenya, Nigeria, and South day Cameroon approximately 4000 years ago. They are Africa. The frequency of the G allele observed in the con- fairly homogenous culturally and reside primarily in an trol population in the present study was 62%. This is in area of South Africa known as the Eastern Cape Prov- contrast with the frequency observed in white popula- ince. A previous study on genetic substructure in South tions from the United States and Italy, Asians from China, African Bantu speakers has found that these groups clus- Japan, and the United States, and Indians from the United ter according to linguistic groupings (Xhosa, Zulu, etc).30 States, which ranged from 78% to 88%. For R141L Exfoliation syndrome is common in this population; a (rs1048661), the frequency that we observed for the G al- recent study conducted in a related tribal grouping re- lele of 88% is similar to the other recent study in black ported a prevalence of 6.6%.26 South African individuals (81%)30 but different from that Our finding in the South African Xhosa population is observed in American white (96%) and Japanese individuals that the AA genotype of the rs3825942 (G153D) SNP is (44%). Because R141L is not present in HapMap and Wil- strongly associated with XFS, which is different from the liams et al30 did not evaluate haplotype data, no compari- GG genotype noted in all other population groups re- son can currently be made for the haplotype frequencies ported to date. This is unexpected, especially in light of of the 2 SNPs identified in this study. the strength of the associations described previously. In Although the allele and genotype frequencies in this addition, in our study the association for G153D is with study were in Hardy-Weinberg equilibrium for the con- the minor allele, which is distinct from all other articles trol groups of both SNPs and the cases in the rs1048661 (Table 3). (R141L) analysis, the allele and genotype frequencies

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rs1048661 (R141L) rs3825942 (G153D)

Risk Risk Study (Location) Allele P Value OR (95% CI) MAF Allele P Value OR (95% CI) MAF Thorleifsson et al8 G 2.3 ϫ 10−2 2.46 (1.91-3.16) Iceland 0.349 (T) G 3.0 ϫ 10−21 20.10 (10.80-37.41) Iceland 0.153 (A) (Iceland/Sweden) Sweden 0.318 (T) Sweden 0.121 (A) Fingert et al13 (USA) G .00004 NA 0.400 (T) G .00030 NA 0.120 (A) Fan et al14 (USA) G .005 1.90 (1.23-2.93) 0.281 (T) G 1.6 ϫ 10−15 20.93 (8.06-54.39) 0.205 (A) Yang et al15 (USA) NA NA NA NA G 4.85 ϫ 10−6 NA 0.147 (A) Aragon-Martin et al16 (USA) G 7.74 ϫ 10−9 0.442 0.297 (T) G 3.10 ϫ 10−17 0.168 0.202 (A) Challa et al17 (USA) G .0222 1.86 (1.10-3.15) 0.335 (T) G .0194 3.05 (1.20-7.76) 0.156 (A) Hewitt et al18 (Australia) G 8.48 ϫ 10−4 1.86 (1.27-2.76) NA G 7.83 ϫ 10−5 3.81 (1.88-9.02) NA Pasutto et al19 G 2.9 ϫ 10−19 2.43 (2.00-2.97) 0.348 (T) G 8.22 ϫ 10−23 4.87 (3.46-6.85) 0.149 (A) (Germany/Italy) Ramprasad et al20 (India) G .156 1.49 (0.89-2.51) 0.270 (T) G .0001 4.17 (1.89-9.18) 0.070 (A) Lee et al21 (China/Singapore) G .142 1.38 (0.91-2.08) 0.444 (T) G .0018 0.97 (1.48-81.49) 0.082 (A) Chen et al22 (China) T 6.95 ϫ 10−11 7.59 (3.87-14.89) 0.484 (G) G 8.00 ϫ 10−4 NA 0.104 (A) Fuse et al23 ( Japan) T 7.7 ϫ 10−18 26.0 (18.3-37.1) 0.493 (G) G 4.1 ϫ 10−4 NA 0.123 (A) Hayashi et al24 ( Japan) T 3.0 ϫ 10−19 99.8 (13.8-722) 0.460 (G) G 1.4 ϫ 10−5 NA 0.143 (A) Ozaki et al26 ( Japan) T 6.41 ϫ 10−48 17.79 (11.03-28.71) 0.497 (G) G 1.30 ϫ 10−11 10.87 (4.59-25.75) 0.137 (A) Williams et al31 (South Africa) G 1.7 ϫ 10−5 23.2 (3.0-177.2) 0.190 (T) A 5.2 ϫ 10−13 0.092 (0.045-0.19) 0.380 (A) Rautenbach et al G .00106 NA 0.117 (T) A Ͻ.00001 9.94 (4.75-20.79) 0.383 (A) (South Africa)a

Abbreviations: CI, confidence interval; MAF, minor allele frequency; NA, data not available; OR, odds ratio. aPresent study.

for the rs3825942 (G153D) were not in Hardy-Wein- either the genomic environment of LOXL1, the molecu- berg equilibrium (P=.00002) for cases with XFS. In lar biological environment of elastin fibril metabolism, light of the fact that the same genotyping platform was or the broader environment in which the pathway func- used in all the genetic analyses, and that similar find- tions act to increase the complexity of the relationship ings have occasionally been described in other case- between LOXL1 and XFS, resulting in these paradoxical control studies of XFS involving these same SNPs20,24 as associations. Progress in understanding this relation- well as in similar studies of other conditions, it is our ship is dependent on further research in all of these opinion that this is a real finding despite deviation from areas. Hardy-Weinberg equilibrium. We are, however, uncer- In summary, the LOXL1 SNPs rs1048661 (R141L) and tain of its implications regarding Hardy-Weinberg as- rs3826942 (G153D) are significantly associated with XFS sumptions in our population. As the sample population in the black South African population. The AA geno- is identical for both SNPs, it is unlikely that this finding type of G153D confers XFS risk in this population, as op- is related in any way to small sample size, inbreeding, or posed to the GG genotype reported in all previously de- assortative mating of any kind but might be a reflection scribed populations. of an association between this marker and disease sus- The fact that the disease-associated haplotype differs ceptibility.32 across various populations (GA in black South African The AA genotype of G153D confers XFS risk in this individuals, TG in Japanese individuals, and GG in white South African population. This observation, together individuals) indicates that these are not the disease- with findings in several Chinese and Japanese popula- causing variants but that they are in linkage disequilib- tions that the TT genotype of the rs1048661 (R141L) rium with the actual pathogenic variants. SNP is strongly associated with XFS contrasts the association of the GG genotype reported in all other North Submitted for Publication: March 14, 2010; final revi- American and European studies as well as our study sion received June 1, 2010; accepted June 7, 2010. (Table 3), suggesting that the original assumption that Correspondence: Robyn M. Rautenbach, MBChB, these 2 SNPs in LOXL1 are causative of XFS is in need of DipOphth(SA), Ophthalmology Department, Tyger- revision. Several possible hypotheses can be proposed berg Academic Hospital, Private Bag X3, Tygerberg 7505, for this finding. The most likely explanation is that, al- South Africa ([email protected]). though a LOXL1 polymorphism is intimately involved Author Contributions: Dr Rautenbach had full access with the pathogenesis of XFS, it is neither the R141L or to all of the data in the study and takes full responsi- G153D but another variant in linkage disequilibrium bility for the integrity of the data and the accuracy of with these SNPs that may not have been included in the the data analysis. original genome-wide association study. Other possible Financial Disclosure: None reported. hypotheses compatible with these findings would as- Funding/Support: This study was supported by re- sume that the R141L and G153D polymorphisms are search funds from the Division of Ophthalmology at Stel- themselves integral to the pathogenesis of XFS but that lenbosch University, Cape Town, South Africa.

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