Visual impairment and progressive phthisis bulbi caused by recessive pathogenic variant in MARK3

Abstract

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2: c.1708C > G: p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.

Figure 1.

MARK3 variant segregation in the Pakistani family pedigree F105 and eye phenotype. Genotypes of the variant (M), also shown by chromatograms (reverse complement), in the pedigree diagram of a consanguineous family of three affected and two unaffected individuals. Photographs of eyes of the affected individuals are shown in the lower panel. Half-filled symbols denote a carrier for the pathogenic variant. Filled symbols denote affected individuals.

Figure 2.

MRI and B-scans of two affected individuals (V: 2 and V: 3). B and C: T1 weighted MRI images of patients V: 2 and V: 3, respectively, arrows indicate the shrunken eye globes; A is the representative MRI image with normal eye globes. E and F are the B-scan images of patients V: 2 and V: 3, respectively, showing abnormal eye globe; D is a representative B-scan of a normal eye.

Figure 3.

Drosophila Par-1 is required for proper eye development and photoreceptor differentiation. (A–C) Adult heads with eyes in which various transgenes were expressed: ey-Gal4 in a y w background (A), ey-Gal4 driving UAS-par-1 RNAi-1 (B), or UAS-par-1 RNAi-2 (C). Expression of either par-1 RNAi-1 (BL#35342) or par-1 RNAi-2 (BL#32410) by ey-Gal4 causes small eyes (B and C). (D–F) ERGs of flies shown above. Arrows indicate the loss of on- and off-transients of ERGs. Quantification of eye sizes (G) (n=10; error bars represent ±SEM, ∗∗∗P<0.001, t-test) (H and I). Quantification of the ERG amplitude (H) and on-transients (I) of ERG traces in (D–F) (n=13; error bars represent ±SEM, ∗∗∗P<0.001, t-test). (J–O) Expression of par-1 RNAi-1 driven by ey-Gal4 significantly reduces the levels of Par-1 in eye discs. (J and K) Eye imaginal disc is stained for Par-1 (red) and the pan-neural marker Elav (white). Par-1 is ubiquitously expressed in the developing eye disc (L). The expression of Par-1 is severely reduced in eye disc of ey > UAS-par-1 RNAi-1 (M). (N and O) Knockdown of Par-1 results in a dramatic reduction in photoreceptor numbers. Elav staining in wild-type control discs (N). Knockdown of Par-1 severely reduces the number the photoreceptors in developing eye disc. Scale bars, 50  μm (J–O).

Figure 4.

Par-1 p.Arg792Gly, the Drosophila mutation homologous to the human MARK3 p.Arg570Gly variant, affects eye development and function. (A) Multiple-sequence alignment of the protein domain of MARK3 that contains the p.Arg570Gly shows that this domain is evolutionarily conserved from flies to human. (B–D) Adult fly heads expressing UAS-lacZ (B), UAS-par-1 WT (C), or UAS-par-1 p.Arg792Gly (D), when driven by ey-Gal4. Expression of Par-1 WT causes a mild defect in eye morphology (C), whereas expression of the p.Arg792Gly protein causes severely reduced eyes that are rough (D). Quantification of the eye sizes (E) (n=10; error bars represent ±SEM, P<0.001, t-test). (F–H) ERGs of flies carrying ey-Gal4 with UAS-lacZ (F), UAS-par-1 WT (G), or UAS-par-1 p.Arg792Gly (H). Quantification of the ERGs shows that expression of the Par-1 p.Arg792Gly mutation causes a near complete loss of ERG amplitude (I) (n=10; error bars represent ±SEM, P<0.001, t-test).