(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date WO 2013/043130 Al 28 March 2013 (28.03.2013) P O P C T
(51) International Patent Classification: Building Level 5, Singapore 16875 1 (SG). KHOR, Chiea- C12Q 1/68 (2006.01) Chuen; Genome Institute of Singapore, 60 Biopolis Street, Genome, Singapore 138672 (SG). (21) International Application Number: PCT/SG2012/00035 1 (74) Agent: CHUNG, Jing Yeng; Marks & Clerk Singapore LLP, Tanjong Pagar, P.O Box 636, Singapore 9108 16 (22) International Filing Date: (SG). 24 September 2012 (24.09.2012) (81) Designated States (unless otherwise indicated, for every (25) Filing Language: English kind of national protection available): AE, AG, AL, AM, (26) Publication Language: English AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, (30) Priority Data: DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, 61/537,904 22 September 201 1 (22.09.201 1) US HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, (71) Applicants: SINGAPORE HEALTH SERVICES PTE KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, LTD [SG/SG]; 31 Third Hospital Avenue, #03-03 Bowyer ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, Block C, Singapore 168753 (SG). AGENCY FOR SCI¬ NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, ENCE, TECHNOLOGY AND RESEARCH [SG/SG]; 1 RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, Fusionopolis Way, #20-10 Connexis, Singapore 138632 TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (SG). ZM, ZW. (72) Inventors: AUNG, Tin; Singapore Eye Research Institute, (84) Designated States (unless otherwise indicated, for every 11 Third Hospital Avenue, SNEC Building Level 5, Singa kind of regional protection available): ARIPO (BW, GH, pore 16875 1 (SG). VITHANA, Eranga; Singapore Eye GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Research Institute, 11 Third Hospital Avenue, SNEC UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ,
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(54) Title: METHOD AND/OR PROBE FOR DETERMINING GLAUCOMA SUSCEPTIBILITY AND/OR PREDICTING/DE TECTING SHALLOW ANTERIOR CHAMBER DEPTH (57) Abstract: The present invention provides a method and/or probe for predicting and/or detecting a shallow anterior chamber depth and/or determining susceptibility to glaucoma in a subject, comprising determining the geno type of the subject for at least one nucleic acid variation in ATP-binding cassette sub-family C member 5 (ABCC5) gene, wherein the nuc leic acid variation is correlated with anterior V chamber depth and/or susceptibility to angle closure glaucoma. - i
Figure 1 WO 2013/043130 Al III III II II II III 11
TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, Published: EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, — with international search report (Art. 21(3)) LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, — with sequence listing part of description (Rule 5.2(a)) GW, ML, MR, NE, SN, TD, TG). Declarations under Rule 4.17: — of inventorship (Rule 4.17(iv)) Method and/or probe for determining glaucoma susceptibility and/or predicting/detecting shallow anterior chamber depth
Field of the invention
The present invention relates to the field of molecular biology, in particular in prediction and/or diagnosis of a condition and/or disease and/or determining susceptibility to the condition and/or disease.
Background of the invention
Glaucoma is known to be a leading cause of irreversible blindness worldwide, characterised by progressive loss of axons in the optic nerve and corresponding visual field damage. Categorized according to the anatomy of the anterior chamber angle, there are two main forms of glaucoma, primary open angle glaucoma (POAG) and primary angle closure glaucoma (PACG). While POAG is the predominant form of glaucoma among Caucasians and Africans, 80% of the estimated 5 million people afflicted with PACG live in Asia. PACG is responsible for substantial blindness in Mongolia, Singapore, China, and India, and it is estimated that PACG blinds proportionately more people than POAG globally.
Both POAG and PACG have indications of being a complex disease with both genetic and environmental factors likely having a role in its etiology. The pathogenesis of PACG is thought to occur through the interplay of multiple anatomical and physiological factors. A major ocular anatomical risk for PACG is a shallow anterior chamber depth of the eye (Lowe 970). However, genes associated with shallow anterior chamber depth, PACG and POAG are largely unknown although a few genes have been reported to have an association with
POAG [Burdon et a/., (201 1); Thorleifsson et al., (2010); Wiggs et a , (201 )]. Summary of the invention
According to a first aspect, the present invention provides a method for predicting and/or detecting a shallow anterior chamber depth and/or determining susceptibility to angle closure glaucoma in a subject, comprising determining the genotype of the subject for at least one nucleic acid variation in ATP-binding cassette sub-family C member 5 (ABCC5) gene, wherein the nucleic acid variation is correlated with anterior chamber depth and/or susceptibility to angle closure glaucoma.
The present invention also provides a probe for use in predicting and/or detecting a shallow anterior chamber depth and/or determining susceptibility to angle closure glaucoma in a subject, wherein the probe is for use in determining the genotype of the subject for at least one nucleic acid variation in ATP binding cassette sub-family C member 5 (ABCC5) gene, wherein the nucleic acid variation is correlated with shallow anterior chamber depth and/or susceptibility to angle closure glaucoma.
According to another aspect, the method and/or probe may be for diagnosing a shallow anterior chamber depth and/or glaucoma in a subject.
Accordingly, the method and/or probe may be used to determine susceptibility to and/or diagnose primary angle closure glaucoma (PACG).
Brief description of the figures
Figure 1 shows the regional association and linkage disequilibrium plot around ABCC5 rs 40 999. The horizontal axis denotes genomic position along Chromosome 3 in absolute base-pairs. The vertical axis shows -Log10 P- values derived from the unguided 2 degrees of freedom genotype. Figure 2 shows the Manhattan plot for genome-wide meta-analysis of anterior chamber depth in three populations (Singapore Malays, Singapore Indians, and Chinese from Beijing) totaling 4,473 individuals.
Figure 3 shows plots of genetic ancestry using principal components for the Singapore GWAS sample (PACG cases and controls) against HapMap reference population panels.
Figures 4A and 4B shows plots of genetic ancestry using principal components for the Singapore PACG cases and controls.
Figure 5 shows the quantile-quantile plot of P-values from the meta-analysis of ACD across the 3 independent sample collections.
Figure 6 shows expression analysis of ABCC5 in human ocular tissues. The 249bp RT-PCR product was observed in cornea (C), sclera (S), retina and retinal pigment epithelium (RPE), iris (I), lens (L), lens capsule (LC) and in the optic nerve (ON). The ubiquitously expressed gene, ACTB was used as the normalizing control. M denotes molecular-weight marker.
Definitions
"Determine" or "determining" means to find out or come to a decision about by investigation, reasoning, or calculation, or to settle or decide from alternatives or possibilities; "determine" or "determining" encompasses the meaning of "predicting" or "assessing".
"Determining the genotype of a subject for a nucleic acid variation" refers to determining the genetic constitution or makeup of the subject for that nucleic acid variation and includes determining the absence or presence of the nucleic acid variation and; where the nucleic acid variation comprises several alternatives, determining the alternative in the subject. "Endophenotype" relates to a hereditary characteristic that is normally associated with some condition (for example, a disease) but is not a direct symptom of that condition. It may also be regarded as an intermediate phenotype of the condition.
"Nucleic acid variation" includes, but is not limited to, a single nucleotide polymorphism (SNP), a double nucleotide polymorphism (DNP), a nucleotide deletion, a nucleotide addition, a nucleic acid amplification, a rearrangement of a nucleic acid sequence or a gene and/or and/or its corresponding transcriptional and/or translational product, and/or alternative splicing of the transcriptional and/or translational product.
A "single polynucleotide polymorphism (SNP)" refers to a DNA and/or RNA sequence variation occurring when a single nucleotide in an organism's DNA sequence differs between members of the species (or between paired chromosomes in the organism).
A "double polynucleotide polymorphism (DNP)" refers to two single polynucleotide polymorphisms, and includes the circumstances when the two SNPs are positioned next to each other, separated by other nucleotides, on different strands of the same nucleic acid molecules, or on different nucleic acid molecules.
A "primer" refers to an oligonucleotide to which deoxyribonucleotides may be added by a DNA polymerase. A single primer may be used to amplify a DNA or RNA region, for example, for sequencing.
A "primer pair" usually comprises a first primer complementary to one strand of a DNA or RNA molecule and a second primer complementary to a second strand of a DNA or RNA molecule, with both primers flanking a target DNA or RNA region, to be amplified by a DNA polymerase. Detailed description of the invention
The present invention relates to a method for predicting and/or detecting a shallow anterior chamber depth and/or determining susceptibility to angle closure glaucoma in a subject, comprising determining the genotype of the subject for at least one nucleic acid variation in ATP-binding cassette sub-family C member 5 (ABCC5) gene, wherein the nucleic acid variation is correlated with susceptibility to angle closure glaucoma and/or shallow anterior chamber depth. In particular, the method is performed on an isolated sample from the subject.
The nucleic acid variation may comprise different alleles of ABCC5. For example, the nucleic acid variation may comprise at least one single nucleotide (SNP) polymorphism of ABCC5. In particular, the nucleic acid variation comprises the rs1401999 single nucleotide polymorphism (SNP).
According to another aspect, the nucleic acid variation comprises a splice variant of ABCC5. Corresponding translational products of such splice variants are also included as part of the invention.
The method of the invention comprises either determining the nucleic acid variation in ABCC5 genomic sequence, and/or its corresponding transcriptional product and/or the corresponding variation in its translational product. The method may detect the nucleic acid variation from genomic DNA, cDNA or mRNA.
According to another aspect, a subject at risk for glaucoma and/or PACG may be selected for the method described herein. Determining the nucleic acid variation in the ABCC5 genomic sequence and/or its corresponding transcriptional product may comprise (i) sequencing to detect ;or (ii) hybridisation with a probe specific to detecting; (iii) PCR using primers specific for detecting, or (iv) restriction fragment length polymorphism specific for detecting; the nucleic acid variation in the ABCC5 gene. Any other suitable analytical procedures may be applied to detect the nucleic acid variation in the ABCC5 gene, for example as discussed in Ragoussis (2009).
The method of the invention may comprise sequencing to detect or hybridising with a probe specific for; PCR using primers specific for ; or restriction fragment length polymorphism specific for detecting the rs1401999 SNP.Determining the variation in the translational product of the ABCC5 gene, may comprise contacting an isolated sample from the subject with antibodies specific to detect the variation in the translational product.
The method of the invention may further comprise a step of administering an effective amount of at least one agent for preventing and/or treating glaucoma and/or PACG to the subject.
According to another aspect of the invention, a method of treating or preventing glaucoma and/or PACG in a subject is provided, said subject having been determined to have at least one nucleic acid variation in ATP binding cassette sub-family C member 5 (ABCC5) gene correlated with anterior chamber depth and/or susceptibility to glaucoma, said method comprising administering to the subject an effective amount of at least one agent for treating glaucoma and/or PACG. Determining the nucleic acid variation in the ABCC5 gene may be by a method according to any aspect of the invention.
The ABCC5 gene is located on chromosome 3q27 (NCBI Reference Sequence
NC_000003.1 1), with the sequence reported at Chromosome 3; NCBI Reference sequence NC_000003.1 1 (183637722.. 183735727, complement)
(SEQ D NO.- 1) There are two main alternative transcripts for ABCC5. The cDNA sequence (NCBI Reference Sequence: NM_005688.2; SEQ ID
NO: 2) is known as variant ( 1) and encodes Mutlidrug resistahce-asociated protein 5, MRP5 (the longer protein isoform ( 1). The protein MRP5 is 1437 amino acids in length and is the longer protein isoform 1 (NCBI Reference Sequence NP_005679.2; SEQ ID NO: 3).
The cDNA sequence with NCBI Reference Sequence: NM_001 023587.1 (SEQ ID NO: 4) is referred to as variant (2) and includes two alternate terminal exons; resulting in a shorter protein with a distinct C-terminus (isoform 2) (NCBI
Reference Sequence NP_001 0 18881 .1 ; SEQ ID NO: 5).
Other transcriptional products and splice variants, for example, as described in NCBI are also included in the invention.
Sequence information for the rs1401999 SNP is provided below. The SNP is indicated in bold.
rs1401999 (SEQ ID NO: 10)
ACTAAACTCC AAACACAGTA ACCTTTATTC TGTTCTTGTC CATTCCAAGT TGCACAAAGT ATTAGGCATG GGGTCATTTC CATTCCTCCT CTCTGAGCCC ACATCACTAA ACCAAATCCA TAGATCTGAT CATTTCCCAC TGCTACCACA GGATAAACAT AAGCTTTTCC ACCTGGTTCT GTAGGACCTC CATATTCTGG CCCTGCCCTA ATCCTGCAAG CTCCTCTCCC ATCTCACTGG TGTCCACAGC CCCTACTCTC AAGCCACACT GGACTTCACA CCATTCCTTA CACCACACTG CAATTTCCCA TTTCTCTACT TTGGTCCACA CTCGTTCCTC CACTTGGAAT GCCCCTCCCC TTGTCCCAAT CGCAGTCACA GTTGTCCCCA TTCAGATAGA TTCAAGTCTT CCATTCTTCA TGAGGCAACA TACACTCCCT GGAAAGCTCA CCCTCCCATT CGCCCTTAGA ACTCCATTAG TACTATACAT GTGATGTGGT S TTGTAAGCCA CATTTGTAAG TGCTTGTAAG AGCATAAAAG CATCGGTCTT CCCTAGAACA CTGTGAGTCC CTTAAGGACA GAGATGATGT CTCTCACATG GTTACAACCT GCACAGGCCC TACCCCTGAG AACTGCCTAT AACAGGGCAC ACAGGGACTC AATAAACAGC CATCAGGGTT CTACCTGAGA TAACAGAAAC TACTTAATGC TCTCCTTTCC AAGTTTCTCT TATGTCTTTT TTTTTTCTTG AGATGGAATC ACGCTCTGTC ACCCAGGTTG GAGTGCAGTG GTGCAATCTT AGCTCACTGC AACCTCTGCC TCCTGGGTTC AAGCGATTCT CCTGCCTCAC CCTCCCAAGT AGCTGGGATT ACAGGCGCCT GCCACCACAC CTGGCTAATT TTTGTATTTT TTTGTAAAGA CGGGGTTTCT CTCTATGTTG CCCAAGCTGG TGTCGAATTC CTGGGCGCAA GCAATCTGTC CACCTCAGCC TCCAAAAATG
S may be Cytosine or Guanine
Information on this SNP may also be obtained from the dbSNP database (http://www.ncbi.nlm.nih/gov/projects/SNP/). The present invention also relates to a probe for use in predicting and/or detecting a shallow anterior chamber depth and/or determining susceptibility to glaucoma in a subject, wherein the probe is for use in determining the genotype of the subject for at least one nucleic acid variation in ATP binding cassette sub¬ family C member 5 (ABCC5) gene.
The probe according to the invention is for use in detecting different alleles of ABCC5 gene. In particular, the probe is for use in detecting a single nucleotide polymorphism in ABCC5. More in particular, the probe is for use in detecting rs1 401 999 single nucleotide polymorphism (SNP).
According to another aspect of the invention, the probe is for use in detecting at least one splice variant of ABCC5. The probe according to any aspect of the invention may be for use in detecting the nucleic acid variation in the ABCC5 gene and/or its corresponding transcriptional product and/or the corresponding variation in its translational product.
The probe may comprise at least one nucleic acid molecule. For example, different nucleic acid molecules hybridizing to different variants of ABCC5 may be used to identify the nucleic acid variation. Such probes may be used to identify different alleles of ABCC5, including but not limited to single nucleotide polymorphisms. The different hybridisation probes may be differentially labelled. Detection of hybridized probes may be by any conventional method, including but not limited to fluorescence-based hybridization assays; chemiluminescence- based hybridization assays; and capture hybridization microtiter assays.
The nucleic acid molecules comprising the probes may also be used as polymerase chain reaction (PCR) primers. PCR may be performed on genomic DNA with PCR primer pairs. Reverse-transcriptase PCR may also be performed. Conventional PCR may be performed or real-time PCR may be performed. Detection of PCR products may be by conventional methods, including but not limited to agarose gel electrophoresis, polyacrylamide gel electrophoresis and capillary electrophoresis. The PCR primers may also be labelled for detection.
A combination of PCR and hybridisation techniques may also be used to identify the nucleic acid variation. Genotyping methods as described in Ragoussis (2009) may also be used for identifying the nucleic acid variation. Alternatively, DNA sequencing may also be used to identify the nucleic acid variation.
According to another aspect, the probe may also comprise an antibody. For example, different antibodies may be used to identify different translational products of the ABCC5 gene using any conventional method. The antibodies may be monoclonal or polyclonal.
Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention. EXAMPLES
Standard molecular biology techniques known in the art and not specifically described were generally followed as described in Sambrook and Russel, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New
York (2001 ).
Example 1 Genotyping
A genome-wide association study (GWAS) was conducted on 935 PACG cases and 952 healthy controls with open angles, all of Singaporean Chinese ancestry. From these, 825 cases and 917 controls passed stringent quality checks. The baseline characteristics of this study cohort and the Chinese and Hong Kong replication study are shown in Table .
Table 1: Baseline characteristics of the PACG cases and controls for the GWAS, replication China and replication Hong Kong study.
A total of 62 SNPs exceeded P < 10 4 for disease association using score- based tests and were brought forward for replication in an additional 988 PACG cases and 1,656 controls from China as well as 246 PACG cases and 225 controls from Hong Kong, all of Han Chinese descent. This list of SNPs was supplemented with an additional 4 SNPs which showed suggestive evidence of association (P < 5 x 0 ) in a meta-analysis of 3 independent GWAS collections (comprising Singaporean Malays, N=1744; Indians, N = 1857 and Beijing Chinese, N=872 for ACD as a quantitative trait (Figure 2). Significant evidence of replication was observed for rs1401999 located within ABCC5 on chromosome 3 , leading to a genome-wide significant observation on meta¬ analysis of data from all samples (Table 2 , N = 2,059 cases and N = 2,798 controls; 2 degrees of freedom unguided genotype test, P = 2.77 x 10 10). Significant deviation from the additive model of association, with the dominant model consistently replicating across all three sample collections was observed (Tables 2 and 3). In contrast, the P-value under the additive model was 1.83 x 10 6, which did not reach genome-wide significance. Consistent evidence of replication was not observed in all other SNPs tested in both the China and Hong Kong sample collections despite having > 90 percent statistical power to validate these associations. Table 2: Association analysis between ABCC5 rs1401999 with PACG
CO c O c >
a : P-value for GG vs. GC + CC under the dominant model
0 m b: Unguided 2 degrees of freedom genotype test; P = 2.78 x 0 MAF: Minor allele frequency m m Table 3 : Genotype analysis of CC rs1401999 and association with PACG. The study is outlined in detail below.
GWAS collection
PACG subjects
935 subjects with PACG were recruited. A case of angle closure was defined as either primary angle closure glaucoma [PACG] or previous acute primary angle closure (APAC). PACG was defined as the presence of appositional angle closure for > 180 degrees with peripheral anterior synechiae and/ or raised intraocular pressure associated with glaucomatous optic neuropathy (defined as loss of neuroretinal rim with a vertical cup: disc ratio of > 0.7 or an inter-eye asymmetry of > 0.2, and/or notching attributable to glaucoma). Previous APAC was defined as the presence of at least two of the following symptoms: ocular or periocular pain, nausea or vomiting or both, and an antecedent history of intermittent blurring of vision with haloes; a presenting intraocular pressure of more than 28 mmHg on Goldmann applanation tonometry; and the presence of at least three of the following signs: conjunctival injection, corneal epithelial edema, mid-dilated unreactive pupil, and shallow anterior chamber (Lee et al., 2007). Patients diagnosed with secondary angle closure (such as neovascular or uveitic glaucoma), patients who had corneal abnormalities that would affect imaging, and patients who had previous laser iridoplasty or intraocular surgery history were excluded. All subjects with angle closure had previously undergone laser peripheral iridotomy (LPI). Genotyping was performed on 935 samples (of which 307 had APAC).
Controls
Controls were ascertained from an on-going population based study of Chinese persons aged 40 years and older [the Singapore Chinese Eye Study (SCES)]. The SCES is a population-based, cross-sectional study of Chinese adults residing in the south-western part of Singapore. The Ministry of Home Affairs provided an initial computer-generated list ethnic Chinese names of adults aged 40-80+ years of age. A final sampling frame of 6,350 ethnic Chinese residents was derived from this list using an age-stratified random sampling strategy. The ongoing SCES began in February, 2009 with an aim to recruit 3,300 (75% response rate) participants (Lavanya et al., 2009). As of March 20 , we recruited and genotyped 952 participants. A control is defined as IOP = 2 1 mm Hg with open angles, healthy optic nerves and normal visual fields, and no previous intraocular surgery.
Chinese replication collection
Angle closure patients including PACG and APAC, were recruited from the Beijing Tongren Hospital, Xingtai Eye Hospital and Anyang Eye Hospital, China. The diagnostic criteria for PACG and APAC were as described above. Controls were recruited from the Handan Eye Study (HES), a population-based study of eye disease in rural Chinese aged 30 years and over. The criteria for selecting controls is also as described above. A total pf 83 SNPs were genotyped for replication with the Sequenom MassArray platform (CapitalBio Corporation, Beijing, China) in 988 PACG cases and 1,656 controls.
Hong Kong replication collection
All subjects of the Hong Kong study population were recruited from the Prince of Wales Hospital and the Hong Kong Eye Hospital, Hong Kong. A total of 246 PACG patients and 225 controls were recruited, all Han Chinese
(Supplementary Table 1). Patients with PACG were defined using the same criteria as that in the Singaporean GWAS collection. In contrast, control subjects were recruited in a hospital-based manner. They were all given complete ocular examinations, and confirmed to have no sign of glaucoma, angle closure or narrow angle, or other major eye diseases except for mild cataract and mild refractive errors. Control subjects were recruited from elderly people aged = 60 years to ensure they were at least free of early-onset major eye diseases. They had IOP < 2 1 mmHg, and had no known family history of glaucoma
Genotyping
For genotyping in both the GWAS discovery and replication stages, genomic DNA samples were extracted from patient and control samples using standard laboratory techniques. Genotyping was performed with lllumina Human610K Quad BeadChips following manufacturer instructions. For the replication stage, SNPs were genotyped with the Sequenom MassArray platform (http://sequenom.com).
Statistical analysis
A selection of stringent quality control (QC) filters were applied to remove poorly performing SNPs and samples using tools implemented in PLINK version 1.7. The QC criteria were as follows: SNPs that had > 5% of missing genotypes, gross departure from Hardy-Weinberg equilibrium (test for HWE showing P < 10 6) or were of minor allele frequency below 1% were excluded from downstream analysis. A total of 485,066 SNPs passed QC and were included for association analysis with PACG disease status. For sample QC, samples with an overall genotyping call rate of < 95% were excluded from analysis. Samples were then subjected to biological relationship verification by using the principle of variability in allele sharing according to the degree of relationship. Identity-by-state (IBS) information was derived by PLINK. From a total of 1,887 samples undergoing GWAS genotyping (935 cases and 952 controls), none had per-sample call-rates of < 95 percent. We found a total of 33 pairs of first degree relatives for all samples passing QC, and from there, the sample with the lower call rate within the pair was removed from downstream analysis. Principle component (PC) analysis was undertaken to account for spurious associations resulting from ancestral differences of individual SNPs. PC plots (Figures 3 and 4) were performed using the R statistical program package (http://r-project.org/). A total of 2 samples had outlying ancestry on PC analysis and were removed from subsequent analysis leaving 1,742 samples (825 cases and 917 controls) for further association analysis.
For the GWAS stage, primary analysis of association with PACG was carried out using a 1 degree of freedom (d.f.) score-based test, which is a trend test incorporating two continuous variables representing the top two principal components of population stratification in order to correct for any residual population substructure. We also performed an accompanying 2 d.f. unguided genotype test for each SNP using logistic regression to verify non-significant departure from the additive model. Analyses in the replication stages were similar to the GWAS stage, conducted primarily with the trend test accompanied by an unguided genotype test to verify non-departure from the additive mode of inheritance. Meta-analysis for genotype-based tests was performed using logistic regression stratified by study collection. The combined P value over all collections for the additive test was generated similarly (based on summing the scores and variances from each stage) as previously described (Kote-Jarai et a/., 201 1). P < 5 x 10 8 was considered as statistically significant.
Statistical power calculations
All power calculations were performed as previously described (Purcell et ai, 2003). Power calculations for the combined replication collection to detect significant replication at P < 0.005 are presented in Table 4. Table 4: Replication study power (based on a combined replication sample of N = 1,234 cases and 1,881 controls) as a function of disease allele frequency and genotypic odds ratio.
These power calculations are based on the ability of the combined replication sample to detect an effect size given the risk allele frequency at P < 0.0005.
Boxes highlighted in bold denote situations where power to detect exceeded 80 percent.
Example 2 Anterior Chamber Depth (ACD)
As anterior chamber depth (ACD) is an anatomical risk factor for primary angle closure glaucoma (PACG), testing for associations between SNP genotypes and ACD, followed by testing for associations between the same SNP genotypes and PACG in the same sample collections will give rise to bias, due to the correlation between ACD and PACG. In light of this, the sample collections undergoing association analysis for the actual disease (PACG; Singapore Chinese, Beijing Chinese, and Hong Kong Chinese) are independent and distinct from the sample collections undergoing association analysis for this quantitative endophenotype (ACD; Singaporean Malays, Singaporean Indians, and an independent collection of Beijing Chinese).
The baseline characteristics of the ACD study cohort are shown in Table 5. Table 5 : Baseline characteristics of sample collections used in quantitative trait analysis for anterior chamber depth (qenotvped individuals passing quality checks from SiMES, SINDI and BES).
Measurement of Anterior Chamber Depth (ACD)
ACD was measured using the lOLMaster (Carl Zeiss Meditec, Dublin, CA). Five readings were obtained and the average computed. The signal-to-noise ratio for all readings were >2.0, which indicate that a clear signal was obtained when performing the measurement. All the readings were within 0.05 mm of the one with the highest signal-to-noise ratio.
Sample collections analyzed for Anterior Chamber Depth
Anterior chamber depth (ACD) measurements were derived from three population based samples: The Singapore Malay Eye Study (SiMES), the Singapore Indian Eye Study (SINDI) and the Beijing Eye Study (BES).
SiMES: The Singapore Malay Eye Study (SiMES) is a population-based, cross- sectional study of 3280 Malay adults aged 40 to 79 years. Details of the SiMES design, sampling plan, and methods have been reported elsewhere (Foong et a/., 2007) In brief, an age-stratified random sampling of all Malay adults, aged 40 to 80 years, residing in 15 residential districts in the south- western part of Singapore was drawn from the computer-generated random list of 16,069 Malay names provided by the Ministry of Home Affairs. A total of 1400 names from each decade of age (40-49, 50-59, 60-69, and 70-79 years), or 5600 names, were selected. Of these, 4168 individuals (74.4%) were determined to be eligible to participate. A person was considered ineligible if he or she had moved from the residential address, had not lived there in the past 6 months, was deceased, or was terminally ill. Of the 4168 eligible individuals, 3280 participants (78.7%) took part in the study. The study was conducted from August, 2004 to June, 2006.
SINDI: As with SiMES, the Singapore Indian Eye Study (SINDI) is a population- based, cross-sectional epidemiological study, but of ethnic Indian adults aged between 40 and 80+ years residing in Singapore. As with SiMES, the Ministry of Home Affairs provided an initial computer-generated list of Indian names derived from'a simple random sampling of all ethnic Indian adults aged 40-80+ years of age residing in 15 residential districts in south-western Singapore. From this list, a final sampling frame of 6,350 ethnic Indian residents was derived using an age-stratified random sampling strategy similar to SiMES. SINDI was conducted from March, 2007 to December, 2009 and recruited 3,400 (75% response rate) participants (Lavanya et a/., 2009).
BES: The Beijing Eye Study is a population-based, cross-sectional study of Chinese adults aged 40+ years and residing in 4 communities in the urban district of Haidian in the North of Central Beijing and in 3 communities in the village area of Yufa of the Daxing District south of Beijing (Zhang et al., 2008). At the time of the first survey in the year 2001 , the 7 communities had a total population of 5324 individuals aged 40 years or older and eligible to take part in the study. In total, 4439 individuals participated in the eye examination (83.4% response rate). In the year 2006, when blood samples were taken, the study was repeated by re-inviting all participants from the survey from 2001 to be re¬ examined with 3251 subjects participating (73.3% response rate).
Analysis of anterior chamber depth
Genome-wide per-cohort and meta-analysis of ACD for all three sample collections was performed using standard procedures as previously described
(Vithana et al., 2001 ; Khor er a/., 201 1). The ACD measurements used in the GWAS excluded any measurements from any eye which were pseudophakic or aphakic. For collections with data from two phakic eyes (SiMES, SINDI), individuals were excluded whose ACD measurements between the two eyes differed more than 0.2mm (which represented the top ~20th percentile of symmetrical data) which gave a good GWAS correlation between the left and the right eye (r2 > 0.95 in both SiMES and SINDI). However, given that BES only had measurements for the right eye, final meta-analysis used ACD measurements taken from the right eye in all three cohorts. A total of 1752, 1860, 872 individuals with complete data for ACD measurements, age and gender were available for SiMES, SINDI and BES individual GWAS. The overall genomic inflation factor for the meta-analysis of the three sample collections was minimal (Age = .036; Figure 5).
Table 6: Quantitative trait analysis between ABCC5 rs1401 999 and anterior chamber depth.
SIMES: Singapore Malay Eye Study SINDI: Singapore Indian Eye Study BES: Beijing Eye Study A 1: Minor allele : Per-allele effect size of ABCC5 rs1 401 999 on anterior chamber depth. SE: Standard error Pgc: Genomic control corrected P-value MAF: Minor allele frequency Example 3 Axial length
Axial length was measured using lOLMaster or A-scan ultrasound biometry.
Table 7 Quantitative trait analysis between ABCC5 rs 40 999 and axial length.
SIMES: Singapore Malay Eye Study SINDI: Singapore Indian Eye Study BES: Beijing Eye Study A1: Minor allele β: Per-allele effect size of ABCC5 rs1401999 on axial length SE: Standard error Pgc: Genomic control corrected P-value MAF: Minor allele frequency
Example 4 Expression analysis
Expression of ABCC5 was assessed by semi-quantitative reverse transcription PCR (RT-PCR) using ABCC5 specific primers [forward 5'- ATTGGCATTGTGGGGCGGAC-3' (SEQ ID NO: 6) and reverse 5'- CCTCTCCAGGGCATCCCAAATC-3' (SEQ ID NO: 7)] on total RNA extracted from a variety of ocular tissues (cornea, sclera, retina and retinal pigment epithelium, iris, lens, lens capsule and optic nerve) with TRIzol® Reagent (Invitrogen, Carlsbad, California) in accordance with the manufacturer's protocol. First-strand cDNA synthesis was performed with Superscript First- Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California). Semi quantitative RT-PCR was performed according to manufacturer's protocol, with the SYBR® Green Master Mix (Invitrogen, Carlsbad, California) using the above ABCC5 primers. The resulting PCR products were separated on a 2% agarose gel and visualized by ethidium bromide staining. The ubiquitously expressed ACTB gene was amplified using specific primers (forward 5'-
CCAACCGCGAGAAGATGA -3"; SEQ ID NO: 8 and reverse 5'- CCAGAGGCGTACAGGGATAG-3'; SEQ D NO: 9) and used as amplification and normalizing control.
ABCC5 was found to be expressed in anterior segment structures relevant to PACG such as the iris and lens (Figure 6).
Discussion
The protein encoded by ABCC5, is a member of the ATP-binding cassette
(ABC) transporter family (Dean and Allikmets 2001 ). ABCC5, also known as multidrug resistance protein 5 (MRP5), has been shown to participate in tissue defense and cellular signal transduction through efflux of anticancer drugs, toxicants and a second messenger cGMP (Jedlitschky et al., 2000; Wijnholds et al., 2000; Pratt et al., 2005). It is expressed in most human tissues, including the cornea (Karla et al., 2009), retinal pigment epithelium and retina of the eye (Stojic et al., 2007).
The exact role of ABCC5 in the eye is not known. The suggestive association between ABCC5 rs 40 999 with a shallower ACD (P = 1.92 x 10 7, β = -0.043 mm per-copy of the minor allele; Table 6) argue favorably for a role in eye growth, particularly that of the anterior segment. Interestingly, a study in zebrafish suggests that ABCC5 may play an active role in eye development through the regulation of intracellular cGMP levels. Zebrafish ABCC5, which shares 73% amino acid sequence identity with human ABCC5, is highly expressed in the lens of the developing eye (Long et al., 201 1). Notably, the blockage of endogenous ABCC5 activity by its dominant-negative was shown to retard development, producing smaller eyes -and thus predispose anatomically to PACG- as well as overall reduction of body length and pigmentation of embryos. In contrast, Abcc5 knockout mice are healthy without any observable physiological dysfunctions or developmental aberrations of the eye (Wijnholds et al., 2000). It is possible that other transporters may compensate the activity of Abcc5 in mice. A developmental role for ABCC5 in mammalian eyes therefore remains to be defined and will require further detailed studies in model organisms.
As rs1401999 is in linkage disequilibrium (LD) with nearby genes PARL,
HTR3D and HTR3C (Figure 1), it is not clear whether the causal alleles or group of alleles influence ABCC5 or any of the neighboring genes to increase susceptibility for PACG. However the clear expression within ocular tissues and a possible role in eye development make ABCC5 the most attractive candidate gene for PACG, within the LD block containing rs1401999. As individuals with a shorter axial length (AL) tend to have smaller eyes and thus higher risk of PACG, the relationship between rs1401999 and AL was also examined. No significant association between rs1401999 and AL in individuals of Chinese descent from Singapore was observed (Table 7). Therefore this data suggest that ABCC5 confer risk via its control of the development of the anterior chamber or its structures such as the lens, and not the entire eye.
The heterogeneity of PACG as a diagnosis (a characteristic shared with primary open angle glaucoma; POAG) also meant that disease-related endophenotypes could be more informative in elucidating true disease genes as the etiological complexity of PACG is reduced and deconstructed. Quantitative phenotypes allow individuals to be ranked along the continuum of risk, providing much greater information than dichotomous measures of affection status and thus aiding disease gene discovery as shown here and elsewhere (Long et al., 20 ; Kathiresan 2008). We note that none of the 62 SNPs associated with PACG per-se at a higher level of significance (P < 10 4 ) could be subsequently replicated, even when statistical power for detection exceeded 90 percent in the combined China and Hong Kong replication collections (Table 4). Conversely, a substantially less significant PACG GWAS signal (P = 0.003) prioritized for follow up genotyping by analysis of ACD showed strong and consistent replication, confirming the validity of this approach in using quantitative endophenotypes in gene discovery for diagnostically heterogenous disorders. Further work will entail systematic and integrative analysis of PACG endophenotypes (in particular ACD and AL) in genome-wide datasets together with PACG patients in order to further identify susceptibility genes. In summary our study identifies a common genetic variant within ABCC5 as being associated with PACG.
As noted above, genes associated with shallow anterior chamber depth and/or primary angle closure glaucoma (PACG) are not known in the art, and the exact role of the ATP binding cassette sub-family C member 5 (ABCC5) gene in the eye is not known in the art. Accordingly, it is not conventional, widely-known or routine to predict and/or detect a shallow anterior chamber depth and/or determine susceptibility to PACG in a subject by determining the genotype of the subject for at least one nucleic acid variation in the ABCC5 gene, in particular by determining that the nucleic acid variation comprises the rs1401999 single nucleotide polymorphism.
Similarly, it is therefore not ponventional, widely-known or routine to use the method of the invention to determine the susceptibility of a subject for glaucoma, in particular PACG. The subject may be selected from a population of subjects known to be at risk for glaucoma and/or PACG, for example due to other risk factors known in the art,
It is also not conventional, widely-known or routine to treat a patient group identified by the method of the invention for glaucoma and/or PACG, using methods of therapy suitable for treating and/or preventing glaucoma and/or PACG, For example, a method of treating or preventing glaucoma and/or PACG in a subject who has been determined to have at least one nucleic acid variation in the ABCC5 gene, comprising administering to the subject an effective amount of at least one substance suitable to treat glaucoma and/or PACG, would not be conventional, widely-known or routine. In particular the at least one nucleic acid variation may comprise the rs 0 999 single nucleotide polymorphism. In the same way, it is not conventional, widely-known or routine to use a probe for determining the genotype of the subject for at least one nucleic acid variation in the ABCC5 gene, in order to predict and/or detect a shallow anterior chamber depth and/or determine susceptibility to angle closure glaucoma in a subject. Any suitable probe for determining nucleic acid variation in the ABCC5 gene may be used, for example if the nucleic acid variation to be determined comprises the rs1401999 single nucleotide polymorphism, any suitable probe for detecting rs1401999 may be used, such as probes described or derivable from sequence information provided at http://www.genecards.org/cgi- bin/carddisp.pl?gene=ABCC5&search=ABCC5&rf=/home/genecards/current/we bsite/carddisp.pl&snp_show_mode=1&snp=210 and/or http://www.ncbi. nlm.nih.gov/SNP/snp_ref.cgi?rs=1 401999 .
References
Burdon, K . P. et al. (201 1) Genome-wide association study identifies susceptibility loci for open angle glaucoma at TMC01 and CDKN2B-AS1 . Nat Genet 43: 574-578.
Cohen, J. C. et ai, (2006) Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 354: 1264-1272.
Dean M and Allikmets R (2001 ) Complete characterization of the human ABC gene family. J Bioenerg Biomembr 33:475-479.
Foong, A. W . et ai.. (2007) Rationale and methodology for a population-based study of eye diseases in Malay people: The Singapore Malay eye study (SiMES). Ophthalmic Epidemiol 14:25-35.
Jedlitschky, G et ai., (2000) The multidrug resistance protein 5 functions as an ATP-dependent export pump for cyclic nucleotides. J Biol Chem 275, 30069- 30074.
Karla, P. K . et ai. (2009) Expression of multidrug resistance associated protein 5 (MRP5) on cornea and its role in drug efflux. J Ocul Pharmacol Ther 25: 121- 132. Kathiresan, S. A (2008) PCSK9 missense variant associated with a reduced risk of early-onset myocardial infarction. N Engl J Med 358: 2299-2300.
Khor, C. C. et al., (201 1) Genome-wide association studies in Asians confirm the involvement of ATOH7 and TGFBR3, and further identify CARD10 as a novel locus influencing optic disc area. Hum Mol Genet 20: 1864-1 872.
Kote-Jarai, Z. et al. (2001 ) Seven prostate cancer susceptibility loci identified by a multi-stage genome-wide association study. Nat Genet 45: 785-791 .
Lavanya, R. et al., (2009) Methodology of the Singapore Indian Chinese Cohort (SICC) eye study: quantifying ethnic variations in the epidemiology of eye diseases in Asians. Ophthalmic Epidemiol 16: 325-336.
Lee, K . Y. et al., (2007) Peripapillary atrophy after acute primary angle closure. Br J Ophthalmol 9 1: 1059-1 061 .
Long, Y. et al., (2010) Molecular analysis, developmental function and heavy metal-induced expression of ABCC5 in zebrafish. Comp Biochem Physiol B Biochem Mol Biol 158: 46-55.
Lowe, R . F (1970) Aetiology of the anatomical basis for primary angle-closure glaucoma. Biometrical comparisons between normal eyes and eyes with primary angle-closure glaucoma. Br J Ophthalmol 54:161-169.
Pratt S. et al., (2005) The multidrug resistance protein 5 (ABCC5) confers resistance to 5-fluorouracil and transports its monophosphorylated metabolites. Mol Cancer Ther 4:855-863.
Purcell, S et al., (2003). Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics 19:149- 150.
Ragoussis J (2009) Genotyping technologies for genetic research. Annu Rev Genomics Hum Genet 10:1 17-33.
Stojic J et al., (2007) Three novel ABCC5 splice variants in human retina and their role as regulators of ABCC5 gene expression. BMC Mol Biol 8:42.
Thorleifsson, G. et al. (2010) Common variants near CAV1 and CAV2 are associated with primary open-angle glaucoma. Nat Genet 42 : 906-909.
Vithana, E. N . et al. (201 1) Collagen-related genes influence the glaucoma risk factor, central corneal thickness. Hum Mol Genet 20: 649-658. Wiggs, J. L . et al. (201 1) Common Variants Near CAV1 and CAV2 are Associated with Primary Open-Angle Glaucoma in Caucasians from the United States. Hum Mol Genet published online 26 August 201 1.
Wijnholds J et al., (2000) Multidrug-resistance protein 5 is a multispecific organic anion transporter able to transport nucleotide analogs. Proc Natl Acad Sci USA 97:7476-7481 .
Zhang, H . et al., (2008)Central corneal thickness in adult Chinese. Association with ocular and general parameters. The Beijing Eye Study. Graefes Arch Clin Exp Ophthalmol 246: 587-592. A method for predicting and/or detecting a shallow anterior chamber depth and/or determining susceptibility to angle closure glaucoma in a subject, comprising determining the genotype of the subject for at least one nucleic acid variation in ATP-binding cassette sub-family C member 5 (ABCC5) gene, wherein the nucleic acid variation is correlated with shallow anterior chamber depth and/or susceptibility to glaucoma.
The method according to claim 1, wherein the glaucoma comprises primary angle closure glaucoma (PACG).
The method according to claim 1 or 2 , wherein the nucleic acid variation comprises different alleles of ABCC5.
The method according to any one of the preceding claims, wherein the nucleic acid variation comprises at least one single nucleotide polymorphism (SNP).
The method according to any one of the preceding claims, wherein the nucleic acid variation comprises the rs1401999 single nucleotide polymorphism (SNP) (SEQ ID NO: 10).
The method according to any one of claims 1 to 3, wherein the nucleic acid variation comprises a splice variant of ABCC5.
The method according to any one of the preceding claims, comprising determining the nucleic acid variation in the ABCC5 genomic sequence, and/or its corresponding transcriptional product and/or the corresponding variation in its translational product.
The method according to claim 7 , wherein the ABCC5 genomic sequence comprises SEQ ID NO: 1. 9 . The method according to claim 7, wherein the cDNA sequence corresponding to the transcriptional product comprises SEQ ID NO: 2 or 4.
10. The method according to claim 7, wherein the translational product comprises SEQ ID NO: 3 or 5 .
11. The method according to any one of the preceding claims, wherein the method is performed on an isolated sample from the subject.
12. A probe for use in predicting and/or detecting a shallow anterior chamber depth and/or determining susceptibility to glaucoma in a subject, wherein the probe is for use in determining the genotype of the subject for at least one nucleic acid variation in ATP binding cassette sub-family C member 5 (ABCC5) gene; wherein the nucleic acid variation is correlated with shallow anterior chamber depth and/or susceptibility to angle closure glaucoma.
13. The probe according to claim 12, wherein the glaucoma comprises primary angle closure glaucoma (PACG).
14. The probe according to claim 12 or 13, for use in detecting different alleles of ABCC5 gene.
15. The probe according to any one of claims 12 to 4 , for use in detecting a single nucleotide polymorphism in ABCC5.
16. The probe according to any one of claims 12 to 15 , for use in detecting rs1401999 single nucleotide polymorphism (SNP) (SEQ ID NO: 10).
7 . The probe according to claim 12 or 13, for use in detecting at least one splice variant of ABCC5. 18. The probe according to any one of claims 12 to 17, for use in detecting at least one nucleic acid variation in the ABCC5 gene and/or its transcriptional product and/or the corresponding variation in its translational product.
19. The probe according to any one of claims 12 to 18, wherein the probe comprises a nucleic acid molecule.
20. The probe according to any one of claims 12 to 19, wherein the probe comprises an antibody.
2 1. A kit comprising at least one probe according to any one of claims 12 to 20.
22. The method according to any one of claims 1 to 11, comprising selecting a subject at risk for glaucoma and/or PACG.
23. The method according to any one of claims 1 to 11 or claim 22, wherein determining the nucleic acid variation in the ABCC5 genomic sequence and/or its corresponding transcriptional product comprises (i) sequencing to detect;(ii) hybridisation with a probe specific to detect; (iii) ;PCR using primers specific for detecting, or (iv) restriction fragment length polymorphism specific for detecting; the nucleic acid variation in the ABCC5 gene.
24. The method according to claim 23, comprising sequencing to detect or hybridising with a probe specific for; PCR using primers specific for ; or restriction fragment length polymorphism specific for detecting the rs1401999 SNP (SEQ ID NO: 10).
25. The method according to any one of claims 7 to 11 or claim 22, wherein determining the variation in the translational product of the ABCC5 gene, may comprise contacting an isolated sample from the subject with antibodies specific to detect the variation in the translational product.
26. The method according to any one of claims 1 to 11 or 22 to 25, further comprising a step of administering an effective amount of at least one agent for preventing and/or treating glaucoma and/or PACG to the subject.
27. A method of treating or preventing glaucoma and/or PACG in a subject who has been determined to have at least one nucleic acid variation in ATP binding cassette sub-family C member 5 (ABCC5) gene correlated with anterior chamber depth and/or susceptibility to glaucoma, comprising administering to the subject an effective amount of at least one agent for treating glaucoma and/or PACG.
28. The method according to claim 27, wherein determining the nucleic acid variation in the ABCC5 gene is by a method according to any one of claims 1 to 11 or 22 to 26.
INTERNATIONAL SEARCH REPORT International application No. PCT/SG2012/000351
A. CLASSIFICATION OF SUBJECT MATTER C12Q 1/68 (2006.01)
According to International Patent Classification (IPC) or to both national classification and IPC B. FIELDS SEARCHED Minimum documentation searched (classification system followed by classification symbols)
Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
Electronic data base consulted during the international search (name of data base and, where practicable, search terms used) WPI, Epodoc, medline, hcaplus, biosis, embase & Keywords: glaucoma, primary angle closure, narrow angle glaucoma, shallow anterior chamber depth, PACG, POAG, ABCC5, ABC33, MOATC, SMRP, rs140 1999, allele, variant, SNP, polymorphism, sequencing, PCR, genotyping, primer, probe and other like terms
GENOMEQUEST: SEQUENCES 1-5 AT 85% identity AND 6, 7 and 10 at 100% identity
C. DOCUMENTS CONSIDERED TO BE RELEVANT
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
Documents are listed in the continuation of Box C
X Further documents are listed in the continuation of Box C X See patent family annex
Special categories of cited documents: document defining the general state of the art which is not "T" later document published after the international filing date or priority date and not in considered to be of particular relevance conflict with the application but cited to understand the principle or theory underlying the invention earlier application or patent but published on or after the "X" document of particular relevance; the claimed invention cannot be considered novel international filing date or cannot be considered to involve an inventive step when the document is taken alone document which may throw doubts on priority claim(s) or "Y" document of particular relevance; the claimed invention cannot be considered to which is cited to establish the publication date of another involve an inventive step when the document is combined with one or more other citation or other special reason (as specified) such documents, such combination being obvious to a person skilled in the art document referring to an oral disclosure, use, exhibition or other means "&" document member of the same patent family document published prior to the international filing date
Date of the actual completion of the international search Date of mailing of the international search report 8 November 2012 0 8 November 2012 Name and mailing address of the ISA/AU Authorised officer
AUSTRALIAN PATENT OFFICE Esther Ng PO BOX 200, WODEN ACT 2606, AUSTRALIA AUSTRALIAN PATENT OFFICE Email address: [email protected] (ISO 9001 Quality Certified Service) Facsimile No.: +61 2 6283 7999 Telephone No. 0262833129
Form PCT/ISA/210 (fifth sheet) (luly 2009) INTERNATIONAL SEARCH REPORT International application No. C (Continuation). DOCUMENTS CONSIDERED TO BE RELEVANT PCT/SG2012/000351
Category* Citation of document, with indication, where appropriate, of the relevant passages Relevant to claim No.
DAZERT, P. et al., 'Expression and localization of the Multidrug Resistance Protein 5 (MRP5/ABCC5), a Cellular Export Pump for cyclic Nucleotides, in Human Heart', 2003, American Journal of Pathology, vol. 163, pages 1567-1577 X See Pages 1568-1569 and Table 1 12-21
WO 2003/044215 A2 (ONCOMEDX, INC.) 30 May 2003 X See Page 2 and Page 7 12-21
WO 1998/046736 A l (MILLENNIUM PHARMACEUTICALS, INC.) 22 October 1998 X See Pages 8-9 and 32-35, SEQ ID NO: 2 shares >99% identity with present SEQ ID 12-21 NO: 3
KARLA, P.K. et al, 'Expression of Multidrug Resistance Associated Protein 5 (MRP5) on Cornea and Its Role in Drug Efflux', 2009, Journal of Ocular Pharmacology and Therapeutics, vol. 25, no. 2, pages 121-132 A See the whole document 1-28
Form PCT/ISA/2 0 (fifth sheet) (July 2009) INTERNATIONAL SEARCH REPORT International application No. PCT/SG2012/000351
Box No. I Nucleotide and/or amino acid sequence(s) (Continuation of item l.c of the first sheet)
1. With regard to any nucleotide and/or amino acid sequence disclosed in the international application, the international search was carried out on the basis of a sequence listing filed or furnished:
a. (means)
I I on paper
I I in electronic form
b. (time)
I I in the international application as filed
I I together with the international application in electronic form
I I subsequently to this Authority for the purposes of search
2. IX I In addition, in the case that more than one version or copy of a sequence listing has been filed or furnished, the required statements that the information in the subsequent or additional copies is identical to that in the application as filed or does not go beyond the application as filed, as appropriate, were furnished.
3. Additional comments:
Form PCT/ISA/210 (second sheet) (July 2009) INTERNATIONAL SEARCH REPORT International application No. Infonriation on patent family members PCT/SG2012/000351 This Annex lists known patent family members relating to the patent documents cited in the above-mentioned international search report. The Australian Patent Office is in no way liable for these particulars which are merely given for the purpose of information.
Patent Document/s Cited in Search Report Patent Family Member/s
Publication Number Publication Date Publication Number Publication Date
WO 2003/04421 5 A2 30 May 2003 AU 2002352799 A l 10 Ju 2003 CA 2467629 A l 30 May 2003 US 2008254457 A l 16 Oct 2008 US 7732141 B2 08 Jun 010 US 2003 148345 A l 07 Aug 2003 US 7767390 B2 03 Aug 2010 WO 03044215 A2 30 May 2003
WO 1998/046736 A l 22 Oct 1998 AU 7125498 A 11Nov 1998 US 5994130 A 30 Nov 1999 US 6077936 A 20 Jun 2000
US 6162616 A 19 Dec 2000
US 6890713 Bl 10 May 2005
US 2005191307 A l 0 1 Sep 2005 US 7785812 B2 31 Aug 2010 US 201 1020341 A l 27 Jan 20 11
US 8287862 B2 16 Oct 2012 WO 9846736 A l 22 Oct 1998
End of Annex
Due to data integration issues this family listing may not include 10 digit Australian applications filed since May 2001 . Form PCT/ISA/2 10 (Family Annex)(My 2009)