AHI1 is required for outer segment development and is a modifier for retinal degeneration in nephronophthisis

Ciliopathies comprise an increasingly diverse group of genetic disorders, united by their connection to primary cilia and/or basal body dysfunction³. Because of the pervasiveness of this organelle, these disorders manifest in numerous organs. Joubert syndrome (JS, i.e. cerebellar hypoplasia), Leber congenital amaurosis (LCA, i.e. congenital retinal blindness) and nephronophthisis (NPHP, i.e. fibrocystic renal disease) primarily affect three organs frequently diseased in ciliopathies, namely cerebellum, retina and kidney. These diseases are highly genetically heterogeneous and are sometimes variable even within single families, undermining the predictive value of diagnostics and genetic counseling. Though many causative genes have been identified for these Mendelian disorders, our current understanding of the genetics and potential mechanisms is insufficient to explain this variability, and suggests involvement of genetic modifiers.

Mutations in AHI1 are identified in 12% of patients with JS and 20% of patients with JS + LCA^1,2,4,5, though AHI1 mutations are not known to cause non-syndromic LCA. AHI1 protein, or jouberin, contains an N-terminal coiled-coil region, seven WD40 repeats and an SH3 domain⁶ and interacts with nephrocystin-1 (NPHP1)⁷, the gene for which is the most commonly mutated in juvenile NPHP^8,9. To study AHI1 in ciliopathogenesis, we generated targeted conditional and null alleles of murine Ahi1, flanking exons 6-7 with loxP sequences (Supplementary Fig. 1a). Homozygous germline mutants were runted and exhibited high mortality (Supplementary Fig. 1c,d). Brain morphology was grossly preserved and neuronal-specific conditional knockouts (Ahi1 Nestin cKO) showed nearly Mendelian ratios at weaning age (data not shown), suggesting effects outside of the nervous system on survival.

Histological analysis of the retina revealed rapid loss of the outer nuclear (photoreceptor) layer with few nuclei remaining by age one month in Ahi1 germline null mice (Ahi1^−/−, Fig. 1a). As early as P10, transmission electron microscopy showed complete absence of both rod and cone outer segments (OS, specialized disk-shaped membranes of photoreceptor cilia, Fig. 1b, and Supplementary Fig. 2a). This well-preceded the initiation of apoptotic cell death, apparent by ~3 weeks of age, as indicated by activated caspase-3 expression (Fig. 1c). Photoreceptor ciliary axonemes were intact and had normal 9+0 microtubule doublet configuration (Fig. 1b, Supplementary Fig. 2b), arguing against a role in axonemal development. Dark-adapted electroretinograms (ERG) from Nestin-Cre conditional mutants (Ahi1 Nestin cKO) and controls preceding cell loss confirmed absence of activity (Fig. 1d). Consistent with its putative role in cilia function, we found Ahi1 was enriched at the connecting cilium and basal body, and overlapped with centrin-2 (centriole/transition zone marker^10,11) transgene expression (Fig. 1e). This expression pattern was reminiscent of ciliary transport molecules such as Ift88¹² and also other ciliopathy-associated molecules like Nphp1¹³. These results reveal specific defects of OS morphogenesis and photoreceptor survival associated with absence of Ahi1.

Rhodopsin (rod opsin and cofactor, retinal), responsible for initiating the first steps in light-dependant signal transduction, is also necessary for OS formation^14-17. Disruptions of genes required for ciliary transport result in mislocalized opsin and OS defects^12,18-20, prompting us to test for opsin localization in Ahi1^−/− retina. We found severely disturbed opsin distribution as early as P10 (Fig. 2a). We quantified this in immunogold-labeled sections, which showed significantly increased inner segment labeling in mutants, both in the cytoplasm (IS) and particularly, along inner segment plasma membranes vs. controls (PM, Fig. 2b and Supplementary Fig. 2c). This difference was ~10-fold for both IS and PM (P= 8.0E-06 and 2.2E-07, respectively). Some other proteins implicated in OS morphogenesis appeared grossly intact (Supplementary Fig. 2d). We conclude opsin is significantly mis-accumulated in Ahi1^−/− mice at an early stage of OS morphogenesis.

To further test for cell-type-specific requirements for Ahi1 in opsin distribution, we used Ahi1^flox/flox mice in a series of retinal in vivo electroporation experiments performed at P0 (Supplementary Fig. 3a). The pCAG-Cre:GFP vector drives Cre:GFP under the constitutive chick beta-actin promoter, and was used with pDsRed-CALNL recombination reporter^21,22. Photoreceptors with evidence of Cre recombination based upon expression of DsRed also showed recapitulation of the opsin redistribution phenotype by one month of age (Supplementary Fig. 3b), indicating a requirement for Ahi1 in photoreceptors. To test for temporal-specific requirements for Ahi1, these experiments were repeated using instead pCAG-ERT2CreERT2, which activates Cre under control of 4-hydroxytamoxifen (4-OHT)²². Following 4-OHT dosage at P14, after the peak of OS development, we did not detect opsin accumulation at 2 and 4 weeks past Cre induction, despite evidence of recombination based upon expression of DsRed (n=2, Supplementary Fig. 3c,d). We conclude Ahi1 displays temporal specificity in its function in photoreceptors, possibly akin to the time dependance reported for ciliary transport machinery in kidney phenotypes²³. These data also suggest other factors might be contributing to OS developmental defects in this model.

Mislocalized opsin is frequently associated with retinal degeneration in animal models^19,24, and has been indicated as a major cause of photoreceptor cell death in the absence of heterotrimeric kinesin-2 function (D. Jimeno, V.S. Lopes, K. Khanobdee, X. Song, B. Chen, S. Nusinowitz, D.S. Williams, unpublished data). Because of the striking accumulation of opsin in Ahi1^−/− mice, we hypothesized this might similarly contribute to cell death in Ahi1^−/− photoreceptors. To test this directly, we reduced opsin levels in Ahi1^−/− mice by introducing a rod opsin null allele¹⁵ into the Ahi1^−/− background. Rho^+/− photoreceptors have approximately 40-50% reduction in opsin content^15,25. Reduction in opsin dosage did not affect OS formation, showing similar absence of OS in Ahi1^−/−Rho^+/− compared to Ahi1^−/− (Supplementary Fig. 4), but significantly delayed the cell loss seen in the Ahi1^−/−. We found nearly complete rescue of cell numbers at three weeks of age (n=3-7, P=0.00175) and partial rescue at one month (Fig. 3, n=3-4, P=0.00613). These data support the hypothesis that abnormal accumulation of opsin contributes to the loss of Ahi1^−/− photoreceptors.

Mutations in NPHP1 cause juvenile nephronophthisis with partially penetrant retinopathy. To study Nphp1 in the mouse, we generated a targeted disruption of Nphp1 by insertion of a neobpA cassette into exon 4, generating a null allele, and confirmed absence of Nphp1 expression (Supplementary Fig. 5a,b,c). The photoreceptors of Nphp1^neo/neo mice (hereafter denoted as Nphp1^−/−) formed OSs but underwent gradual retinal degeneration, slightly evident at 2 months (Supplementary Fig. 5d). This was milder than an independently targeted Nphp1 mutant reported to show failure of OS formation followed by retinal degeneration²⁶. Since both Nphp1 alleles are predicted to function as nulls, this difference in severity of retinal phenotypes suggests the influence of genetic modifiers.

To test whether genetic interaction of Ahi1 and Nphp1 can influence phenotypic expression in vivo, we examined double mutant combinations of Ahi1 and Nphp1, ranging from single heterozygotes to knockout; heterozygote combinations, for outer nuclear layer (ONL) thickness and for mislocalized opsin. To efficiently obtain genotypes homozygous null for Ahi1, we used the Ahi1^flox allele and the distal (peripheral) retina-specific Pax6α-Cre transgene²⁷ to circumvent the lethality issues associated with Ahi1^−/−. We observed trends of increasing severity of cell loss and opsin redistribution with additional deleterious alleles, with the Ahi1 null allele showing a stronger effect (Fig. 4a). Specifically we found significantly decreased ONL thickness and increased ONL-localized opsin when we compared Nphp1^−/−Ahi1^+/− against Nphp1^−/− and Ahi1^+/− controls at P21 (Fig. 4b). The data suggest dosage sensitive genetic interactions between Ahi1 and Nphp1 in retinal development.

Based on the pronounced retinopathy in Ahi1^−/− mice, we next tested whether AHI1 was mutated in humans with isolated LCA. In a screen of AHI1 coding exons from well-characterized LCA patients (US, Canada, Netherlands, Spain, prescreened for known genes; n=176), no homozygous or compound heterozygous deleterious changes were identified, but one variant, p.R830W/c.C2488T, heterozygous in 9 independent cases, was of particular interest, though not significantly enriched (data not shown). In addition to conservation of p.R830 throughout vertebrate homologs, a change from arginine to tryptophan (from a polar basic residue to a nonpolar hydrophobic residue) is predicted to be damaging (Polyphen²⁸, SNPs3D²⁹). This coupled with its position within the WD40 repeat domain (between two blades of its predicted propeller structure, Supplementary Fig. 6a,b) suggests this mutation may interfere with the function of AHI1, perhaps in protein-protein interactions.

To test whether this change altered sedimentation of AHI1 complexes in vitro, we expressed GFP-AHI1^R830W, GFP-AHI1^WT or GFP-EV (empty vector) in HEK293T cells with untagged human rhodopsin³⁰ (RHO) and assayed lysates by sucrose density gradients (Supplementary Fig. 6c). We identified two distinct sedimentation peaks containing AHI1 which appeared at lower intensity for GFP-AHI1^R830W transfected cells, suggesting underrepresentation of p.R830W in complexes, despite comparable total expression levels of p.R830W in HEK293T cells (Supplementary Fig. 6d). Furthermore, opsin was shifted towards lower density complexes in cells expressing p.R830W vs. those expressing wildtype AHI1 or empty vector. This supports potentially hypomorphic effects of p.R830W, perhaps in complex stability or formation. This variant has been mentioned as a polymorphism in several AHI1 studies^1,2,31,32, but the effect of this change on retinal disease has not been explored. Due to the genetic interaction of Ahi1 and Nphp1 in mouse, and the variable association of retinal degeneration (RD) with nephronophthisis (Senior-Løken syndrome, SLSN³³), we hypothesized that AHI1 p.R830W might contribute to the risk of retinopathy in nephronophthisis patients.

We thus genotyped 153 independent nephronophthisis cases from Italy (of which 16 have RD), and 306 ethnically-matched healthy controls for the p.R830W (c.C2488T) change. We found the T allele frequency was significantly higher in patients with NPHP+RD versus NPHP patients excluded for RD (25% vs. 1.8%, P=5.36E-06, Table 1) and versus controls (25% vs. 2.8%, P=9.03E-06, Table 1). This translated to a relative risk of 7.5 (95% CI 4.0-11.2) associated with AHI1 p.R830W for retinal degeneration within NPHP patients. To determine if this association depended upon the primary gene mutation, we repeated this analysis after subdividing cases into those with NPHP1 mutations (either homozygous deletion or compound heterozygous mutation) vs. those without NPHP1 mutations. p.R830W continued to be significantly associated with NPHP+RD irrespective of the primary mutation (Table 1).

To test for population stratification, we also examined transmission of p.R830W in this cohort³⁴ by genotyping parents of 117 available trios. We found 17 informative trios where one parent was heterozygous for c.C2488T. Although this sample size was too limited for full TDT analysis, we found the T allele was over-transmitted to NPHP+RD patients. Specifically, T was transmitted 100% of the time to NPHP+RD patients (n=7, P<0.01, chi-squared), but only 50% of the time to NPHP patients without RD (n=10, no significant difference from the null hypothesis). This association was apparent when evaluating single families; for example, in one family with three NPHP affecteds³³, p.R830W segregated only with the sibling displaying absent ERG response, while the others retained visual function. Our results support the role of AHI1 as a modifier of retinal degeneration in the context of mutations leading to NPHP.

That this AHI1 allele is associated with retinal disease in as much as half of SLSN patients in this Italian population, suggests other rarer mutations of AHI1 may behave similarly. Results from the mouse model support a role for Ahi1 in cilia-associated trafficking mechanisms, consistent with those of a recent study describing Ahi1 in cultured cells³⁵. We also identify a new role for Ahi1 in photoreceptor development and demonstrate the utility of mouse genetic interaction in identifying loci conferring high risk alleles for disease. A similar effect was presented with one variant of RPGRIP1L³⁶, supporting the role of modifiers in phenotypic expressivity of ciliopathies. Given the complexity of ciliopathy phenotypes, it is likely other variable phenotypes could also be explained by modifiers. Intriguingly, a population of JS patients (n=155) did not reveal a significant difference in frequency of p.R830W although it was slightly higher relative to controls. These patients were ascertained for phenotypes associated with a specific constellation of midbrain-hindbrain malformations, but also variably present with renal and/or retinal dysfunction. Lack of significant association within JS may reflect more complex interactions at the molecular level, requiring specific disruptions to alter phenotypic expression. Future screenings at other loci may help elucidate these distinct but overlapping mechanisms. Our example within the ciliopathies shows that mutational analysis of other causative genes from the broader clinical spectrum can yield substantial insight into the genetics and pathogenesis of heterogeneous syndromic disorders.