Mapping of the disease locus and identification of ADAMTS10 as a candidate gene in a canine model of primary open angle glaucoma

Abstract

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide, with elevated intraocular pressure as an important risk factor. Increased resistance to outflow of aqueous humor through the trabecular meshwork causes elevated intraocular pressure, but the specific mechanisms are unknown. In this study, we used genome-wide SNP arrays to map the disease gene in a colony of Beagle dogs with inherited POAG to within a single 4 Mb locus on canine chromosome 20. The Beagle POAG locus is syntenic to a previously mapped human quantitative trait locus for intraocular pressure on human chromosome 19. Sequence capture and next-generation sequencing of the entire canine POAG locus revealed a total of 2,692 SNPs segregating with disease. Of the disease-segregating SNPs, 54 were within exons, 8 of which result in amino acid substitutions. The strongest candidate variant causes a glycine to arginine substitution in a highly conserved region of the metalloproteinase ADAMTS10. Western blotting revealed ADAMTS10 protein is preferentially expressed in the trabecular meshwork, supporting an effect of the variant specific to aqueous humor outflow. The Gly661Arg variant in ADAMTS10 found in the POAG Beagles suggests that altered processing of extracellular matrix and/or defects in microfibril structure or function may be involved in raising intraocular pressure, offering specific biochemical targets for future research and treatment strategies.

Figure 1. Pedigree of the POAG Beagle colony.

The most recent generations of Beagles are shown, including the 30 dogs used for SNP genotyping (dogs 1–31, except dog 6). Prior to introduction of two unrelated normal Beagles (dogs 6 and 9), the colony had been maintained by mating affected to affected dogs for 5 generations, 3 of which are shown. Boxes: males, circles: females, filled symbols: POAG-affected, open symbols: not affected, open symbols with line from upper right to lower left: carriers, blue slash: sample not available.

Figure 2. Mapping the POAG locus using the zygosity criterion.

Genome-wide SNP data was evaluated to identify SNPs that satisfy the zygosity criterion, defined as being homozygous for all 19 POAG-affected dogs and heterozygous for all 10 carrier dogs. Calculating the percentage of carrier dogs heterozygous for each SNP revealed that only chromosome 20 contained SNPs for which all carriers (100%) were heterozygous (A). Within chromosome 20, the SNPs heterozygous for all carriers (orange and green symbols) form a contiguous block of 41 SNPs, within which 27 contiguous SNPs satisfy the zygosity criterion (green symbols) (B). Haplotype analysis revealed informative recombinations within the pedigree that defined a 4 Mb disease locus corresponding to the 27 SNPs satisfying the zygosity criterion (C). The location and genotypes of the SNPs satisfying the zygosity criterion are shown in Figure S1.

Figure 3. Genome-wide linkage analysis of SNP genotyping data.

Calculation of two-point LOD scores revealed regions of interest, defined as having LOD score >2, on chromosomes 5, 15 and 20 (A). Two-point (black lines) and follow-up multipoint (red lines) linkage analyses are shown for chromosomes 5, 15 and 20 (B–D). Multipoint linkage analysis reduced the LOD score of the chromosome 5 region of interest to below 1 (B). Multipoint LOD scores for the two regions of interest (R1 and R2) of chromosome 15 were similar or higher than the two-point scores (C). The region on the distal end of chromosome 20 with two-point LOD score >2 had multipoint score of 2.70, and corresponds to the 4 Mb locus identified by the zygosity criterion.

Figure 4. Haplotype analysis of chromosome 15 regions R1 and R2.

Although region R1 (A) and R2 (B) had multipoint LOD scores >2, as shown in Figure 3C, their inheritance patterns were discordant with disease status, ruling out these regions as containing the disease allele. The minimal informative pedigree is shown, with dog numbers and symbols corresponding to the pedigree shown in Figure 1. The chromosomal positions for the SNPs defining the haplotypes are shown in Figure S2.

Figure 5. The canine POAG locus is syntenic with a human quantitative trait locus for intraocular pressure.

The 4 Mb POAG locus (solid blue rectangle) found in the Beagle colony maps to a 6.5 Mb region (solid red rectangle) on the short arm of human chromosome 19, and within a 20 cM quantitative trait locus for intraocular pressure in humans located near microsatellite marker D19S586 (purple symbol) (A). The number and order of genes within the canine POAG locus and the syntenic region of human chromosome 19 are well conserved (B). Canine genes, blue circles, human genes, red circles. Orthologous genes are connected by gray lines. The canine chromosome 20 locus extends from base pair positions 55,881,144 to 59,844,869, corresponding to human chromosome 19 base pair positions 2,389,784 to 8,841,863. Direction of increasing base pair number of the reference sequence is indicated by red and blue arrows (B). At the centromeric end of the canine locus, a 0.3 Mb portion of the locus is inverted with respect to the human chromosome (B). The figure is drawn to scale, with scale bars of 5 Mb (A) and 0.5 Mb (B) shown.

Figure 6. The 56097365 G->A variant is within a highly conserved region of ADAMTS10.

The G->A variant identified by Illumina sequencing was confirmed by Sanger sequencing of normal, affected and carrier dogs from the POAG Beagle colony (A). The variant causes a glycine residue at amino acid position 661 (red) to be changed to an arginine within exon 17 of ADAMTS10 (B). This glycine is conserved in the sequence of 38 vertebrate species (Human, Chimp, Gorilla, Orangutan, Rhesus Monkey, Baboon, Marmoset, Tarsier, Mouse lemur, Bushbaby, Mouse, Rat, Kangaroo rat, Guinea pig, Squirrel, Rabbit, Dolphin, Cow, Horse, Cat, Dog, Megabat, Hedgehog, Elephant, Tenrec, Armadilo, Wallaby, Opossum, Platypus, Chicken, Zebra finch, Xenopus tropicalis, Tetraodon, Fugu, Stickleback, Medaka, Zebra fish, and Lamprey), 7 of which are shown aligned (B). The Gly661Arg variant occurs within the cysteine-rich domain of ADAMTS10 (C).

Figure 7. ADAMTS10 protein is highly expressed in the trabecular meshwork.

Western blotting using an anti-ADAMTS10 (A and B) or anti-GAPDH (C and D) antibody was performed on cell lysates (A and C) or protein extracts from normal canine eye tissues (B and D). For cell lysates, 5 µg total protein from cells transfected with empty vector (Vector), or a vector containing an ADAMTS10 construct (ADAMTS10) were loaded in each lane (A and C). For eye tissue protein extracts, 10 µg total protein were loaded in each lane (B and D). TM: trabecular meshwork, CB: ciliary body, ON: optic nerve. Molecular weight marker (mw) is shown with molecular weights in kDa.

Figure 8. Structural modeling predicts disruption of the normal protein fold by the Gly661Arg variant of ADAMTS10.

The structure of normal (A and B) and Gly661Arg mutated (C) ADAMTS10 was predicted by homology modeling using the amino acid sequence of ADAMTS10 and the crystal structure of ADAMTS13 , which includes the disintegrin-like domain (D), the thrombospondin-1 type-1 repeat domain (T₁), the amino-terminal portion of the cysteine-rich domain (C_A), the carboxy-terminal portion of the cysteine-rich domain (C_B) and the spacer domain (S). Rotated views (90°) of the entire predicted structure for ADAMTS10 are shown, with the portion of backbone corresponding to Gly661 in the C_A domain colored black (A). The boxed portion in A is expanded to show the substitution site for normal (B) and mutated (C) ADAMTS10, with selected amino acid side chains shown.

Figure 9. Gly661Arg-mutated ADAMTS10 decays more rapidly than does normal ADAMTS10.

In vitro transcribed normal (A and blue diamonds in C) and Gly661Arg mutated (B and red squares in C) ADAMTS10 labeled with biotinylated lysine residues was incubated in aqueous humor for various times at 37°C. A band of ∼130 kDa was detected by Western blotting with fluorescently labeled streptavidin, as shown in a representative experiment for both normal (A) and mutated (B) ADAMTS10 (incubation times, in minutes, shown below lanes). The slopes of the lines fit to data from all four experiments combined by normalizing band intensities to the initial time point (C) were significantly different (p<0.001) and correspond to half lives of 255.8 min. for mutated (red squares and red line) and 636.9 min. for normal ADAMTS10 (blue diamonds and blue line) (C). Molecular weight markers are shown in the left-most lanes of the blots (A and B) with mw in kDa indicated.