Mutations in LRPAP1 are associated with severe myopia in humans

Abstract

Myopia is an extremely common eye disorder but the pathogenesis of its isolated form, which accounts for the overwhelming majority of cases, remains poorly understood. There is strong evidence for genetic predisposition to myopia, but determining myopia genetic risk factors has been difficult to achieve. We have identified Mendelian forms of myopia in four consanguineous families and implemented exome/autozygome analysis to identify homozygous truncating variants in LRPAP1 and CTSH as the likely causal mutations. LRPAP1 encodes a chaperone of LRP1, which is known to influence TGF-β activity. Interestingly, we observed marked deficiency of LRP1 and upregulation of TGF-β in cells from affected individuals, the latter being consistent with available data on the role of TGF-β in the remodeling of the sclera in myopia and the high frequency of myopia in individuals with Marfan syndrome who characteristically have upregulation of TGF-β signaling. CTSH, on the other hand, encodes a protease and we show that deficiency of the murine ortholog results in markedly abnormal globes consistent with the observed human phenotype. Our data highlight a role for LRPAP1 and CTSH in myopia genetics and demonstrate the power of Mendelian forms in illuminating new molecular mechanisms that may be relevant to common phenotypes.

Figure 1

Identification of LRPAP1 Mutations in a Mendelian Form of Myopia (A) Pedigrees of three consanguineous families in which extreme myopia appears to follow an autosomal-recessive mode of inheritance. (B) Autozygome analysis shows a single block of autozygosity (boxed in red, the coordinates are 1,725,469 to 4,136,325) on chromosome 4 that is exclusively shared by the affected members of the three families (columns represent individual cases and rows represent individual SNP calls; black is homozygous and yellow is heterozygous). (C) Linkage analysis shows one peak on chromosome 4 with a LOD score of 7. (D) Schematic of LRPAP1 and the protein it encodes with the sites of the two truncating mutations shown. DNA chromatograms are shown for the mutations.

Figure 2

Identification of a CTSH Mutation in a Mendelian Form of Myopia (A) Pedigree of a consanguineous family with one child with extreme myopia. (B) Autozygome analysis shows several blocks of autozygosity but one block is shown that harbors CTSH and next to it is a schematic of the filtration strategy used to highlight the CTSH mutation. (C) Schematic of CTSH and the protein it encodes with the sites of the two truncating mutations shown. DNA chromatograms for the reported mutation are shown.

Figure 3

LRPAP1 and CTSH Mutations Are Probably Loss of Function (A) Immunoblot analysis with LRPAP1 antibody shows nearly complete loss of LRPAP1 in the index of each of families 1–3 compared to controls. (B) RT-PCR of two cDNA fragments from CTSH (Rx1 and Rx2) shows severe instability of the mutant transcript in the index in family 4 compared to control.

Figure 4

LRPAP1 Deficiency Is Associated with Severe Depletion of LRP1 and Upregulation of TGFB1 Top: immunoblot analysis with LRP1 antibody showing marked reduction of the band corresponding to LRP1 in affected individuals compared to controls (GAPDH is shown for loading control). Bottom: immunoblot analysis with TGF-β antibody showing >2-fold increase in TGF-β in affected individuals compared to controls (GAPDH is shown for loading control).

Figure 5

CTSH Deficiency in Mouse Results in Abnormal Globe Development A panel of comparable eye H&E-stained sections from Ctsh^−/− and their wild-type littermate (four animals are shown, two eyes each for a total of eight sections). Note the grossly abnormal globes in the knockout mouse that assume a “<” shape compared to the rounded appearance in controls, most probably indicative of elongated axial length.