Morgagnian cataract resulting from a naturally occurring nonsense mutation elucidates a role of CPAMD8 in mammalian lens development

Abstract

To investigate the genetic basis of hereditary lens opacities we analyzed 31 cases of bilateral congenital cataract in Red Holstein Friesian cattle. A genome-wide association study revealed a significant association on bovine chromosome 7 at positions 6,166,179 and 12,429,691. Whole genome re-sequencing of one case and four relatives showed a nonsense mutation (g.5995966C>T) in the PZP-like, alpha-2-macroglobulin domain containing 8 (CPAMD8) gene leading to a premature stop codon (CPAMD8 p.Gln74*) associated with cataract development in cattle. With immunohistochemistry we confirmed a physiological expression of CPAMD8 in the ciliary body epithelium of the eye in unaffected cattle, while the protein was not detectable in the ciliary body of cattle with cataracts. RNA expression of CPAMD8 was detected in healthy adult, fetal and cataractous lenses.

Fig 1. Congenital cataract in Red Holstein Friesian cattle.

(A) Bilateral mature cataract formation, (B) sagittal cut through the eyeball (formalin fixed) with cataractous lens (animal #224) clearly showing the irregular lens surface. (C) to (E) are pictures of different stages of the degenerative lenses in situ taken during the clinical examinations: (C) left eye of animal #227 at 13 months, mature cataract, (D) right eye of animal #489 at 31.5 months, mature to hypermature cataract, (E) left eye of animal #908 at 30 months, mature to hypermature cataract. Iris abnormalities as observed by Cheong et al. (2016) in human ASD patients showing cataracts were not detected in bovine cataract cases. (F) to (I) show the histopathological changes in hematoxylin/eosin stained sections (bars = 100μm): (F) lens equatorial line in a bovine healthy adult lens where the anterior epithelium (arrow) under the capsule is clearly visible (control animal). During cataract formation the capsule thickens, the cuboidal epithelium (G, arrow, animal #224, left eye) disintegrates and over time vanishes completely (H, animal #908, left eye). The loss of lens fibrils and presence of Morgagnian globules indicate the presence of a hypermature cataract (G, I, animal #224). Fig 1A was digitally improved and cropped using GIMP 2.8.18.

Fig 2. Origin and transmission of cataract in Red Holstein Friesian cattle.

The pedigree depicts the ancestry of the affected individuals (grey, purple and blue symbols). Two cases are not displayed in the family tree. Neither maternal nor paternal lines were clearly documented. Animal #183 was also genotyped T/T for the variant g.5995966C>T in CPAMD8. The origin of the cataract-causing mutation was traced back to founder sire #780 (yellow symbol). The dam´s sires in 20 of 31 cases were sires #053, #890, #977, marked with brown symbols. The red symbol shows sire #870, father of 26 of 31 cases. This animal (#870) was also used for whole genome re-sequencing, as the green (mother and maternal grandmother of one case) and purple (cattle with congenital cataract) marked cattle. The blue and purple symbols mark the four dissected cases. Family tree was created using Pedigraph [22].

Fig 3. Results of genome-wide association study (GWAS) using SNP data of 26 cases and 88 Holstein control animals.

(A) Manhattan plot (generated using Haploview [23]) and calculated -log₁₀ p values showing an association with the cataract phenotype on BTA7, (B) Detailed view of association on BTA7 with -log₁₀ Bonferroni-adjusted p values. Red symbols showing associations of 5.44x10^-30 at position 6,166,179 and 1.85x10^-32 at position 12,429,691, (C) Region of extended homozygosity on BTA7 in cataract cases (grey symbol). CPAMD8 (5,995,747 to 6,095,877) is located at the proximal end of the 4.7 Mb region of extended homozygosity from 5,639,104 to 10,406,009.

Fig 4. Loss of CPAMD8 expression in cataract cases.

Overview of cornea and ciliary body (A, H&E staining). Physiologically, CPAMD8 is detectable by immunohistochemical staining in the epithelium of the ciliary body as cytoplasmic and vesicular staining in light brown (adult control sample) (B). In the cataract ciliary body epithelium, no CPAMD8 immunoreaction is detectable (C); CPAMD8 antibody; visualization by AEC.