Functional analyses of variants reveal a significant role for dominant negative and common alleles in oligogenic Bardet-Biedl syndrome

Abstract

Technological advances hold the promise of rapidly catalyzing the discovery of pathogenic variants for genetic disease. However, this possibility is tempered by limitations in interpreting the functional consequences of genetic variation at candidate loci. Here, we present a systematic approach, grounded on physiologically relevant assays, to evaluate the mutational content (125 alleles) of the 14 genes associated with Bardet-Biedl syndrome (BBS). A combination of in vivo assays with subsequent in vitro validation suggests that a significant fraction of BBS-associated mutations have a dominant-negative mode of action. Moreover, we find that a subset of common alleles, previously considered to be benign, are, in fact, detrimental to protein function and can interact with strong rare alleles to modulate disease presentation. These data represent a comprehensive evaluation of genetic load in a multilocus disease. Importantly, superimposition of these results to human genetics data suggests a previously underappreciated complexity in disease architecture that might be shared among diverse clinical phenotypes.

Fig. 1.

Suppression of BBS4 produces specific defects. (A) Injection of MOs against individual bbs genes (bbs4 shown here) into zebrafish embryos produces gastrulation defects, including a short body axis, a widened and kinked notochord (*), and broadened somites (arrowhead), which can be rescued by WT human mRNA. Coinjections of mutant mRNAs produce a spectrum of defects. (B) Whereas WT RNA rescues the MO phenotypes (WT RNA), coinjection of hypomorphic mutations (N165H) partially rescues the phenotype; however, null mutations (D102G, N274H, and M472V in BBS4) do not rescue, and dominant-negative mutations (L327P) exacerbate the phenotype and produce defects by injection of RNA alone.

Fig. 2.

In vitro validation of mutation effects. (A–C) Localization of myc-tagged WT BBS4 protein (red) in IMCD3 cells shows presence at the basal body, as indicated by colocalization with γ-tubulin and acetylated α-tubulin (green). (D–F) Introduction of an artificial N165F mutation in BBS4 has no observable effect on localization. Expression of the D102G mutant, characterized as null by in vivo scoring, is undetectable 2 d after transfection (G–I), whereas the hypomorphic N165H variant is expressed but mislocalized (J–L). The dominant-negative mutation L327P results in mislocalization in postmitotic cells (M–O) but not in dividing cells (P–R). (S) Expression of MYC-tagged constructs 4 d after transfection is undetectable for null mutations (D102G, N274H, and N165H), although RT-PCR showed message in each case.

Fig. 3.

The dominant-negative effect of BBS4 L327P. (A) Coinjection of 3 ng of bbs4 MO and 100 pg of WT human mRNA rescues the morphant phenotype, and coinjection of 100 pg of L237P bearing BBS4 mRNA produces defects similar to, or more severe than, MO alone. Introduction of equimolar amounts (50 pg each), however, of both WT and mutant mRNA attenuates the ability of 50 pg of WT mRNA alone to rescue partially the morphant phenotype. (B) Injection of a suboptimal dose (10 pg) of L327P mRNA produces significant phenotypes. (C) Overexpression of the dominant-negative BBS4 mutation L327P but not of the loss-of-function alleles activated canonical Wnt signaling by the Super TOP-Flash luciferase assay in a manner similar to BBS4 suppression. Overexpression of L327P mRNA at a high concentration (300 pg) results in dorsalized embryos and double axes (arrows) (D) and ectopic chordin expression (E). (F) In the context of oligogenic inheritance, the primary (recessive) locus has a significantly increased likelihood of being hypomorphic (P < 0.0001), whereas the epistatic locus is more likely to be severe, with significant enrichment of dominant-negative lesions (P = 0.0004).

Fig. 4.

Reevaluation of genetic data using in vivo scoring. Effects of mutations based on in vivo analyses suggest that the severity of BBS mutations is consistent with segregation of disease in families with BBS. (A) Although the heterozygous nonsense BBS1 mutation Q128X present in the individual who has BBS in family AR186 is functionally null (B and C), its presence does not fully account for the disease. (B and C) Affected individual, however, is also homozygous for the BBS2 polymorphism, I123V, predicted to be a mild hypomorph. (D) Injection of either bbs1 or bbs2 MO alone at suboptimal concentrations results in mildly affected embryos. Coinjection of both MOs together produces severely affected embryos with phenotypes not seen by injection of either MO alone, including near-complete loss of somite definition (labeled by myoD) and loss of eye field (labeled by pax2).