De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features

Abstract

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

Keywords: Drosophila; FRYL; developmental delay; furry; intellectual disability; rare disease.

Figure 1

Locations of predicted LoF and missense FRYL variants identified in this study (A) Schematic of human FRYL and its alignment to human FRY and fly Fry. The Fry N-terminal (Pfam: #14222), cell morphogenesis central (Pfam: #PF14225), and cell morphogenesis C-terminal domains (Pfam: #PF14228) are identified in the Pfam project. The C-terminal leucine zipper and coiled-coil motifs are predicted by Goto et al., which are only present in human FRY and FRYL but not fly Fry. Five FRYL missense variants identified in this study are shown above the protein, and nine predicted LoF variants are shown below the protein. (B) The conservation of the five amino acid residues affected by the FRYL missense variants. The “--” symbols indicate that no aligned amino acid residue is found in the corresponding protein. All the residues are conserved in the Fryl proteins across vertebrate species as well as human FRY. In Drosophila, which is used as model organism in this study, four residues are conserved while the Arg110 residue is not. The variants identified in the five affected individuals correspond to amino acid changes, which are shown on the bottom row.

Figure 2

Dysmorphic facial features of individuals with heterozygous variants in FRYL (A and B) Facial images of individual 1 showing bitemporal narrowing, tall forehead, hypertelorism, epicanthal folds, upslanting palpebral fissures, flat nasal bridge with short upturned nose with bulbous tip, long deeply grooved philtrum, and cleft chin. (C and D) Facial images of individual 6 showing hypertelorism, epicanthal folds, flat nasal bridge, and ear pit (indicated by the arrow).

Figure 3

Fry is essential for fly development, and the loss of fry causes morphological defects in the wings and compound eyes (A) Schematic of the generation of the fry^T2A-GAL4 allele. The fry^T2A-GAL4 allele is predicted to be a severe LoF allele. It also induces the expression of GAL4 protein under the control of the endogenous regulatory elements of fry, which can be used to determine the expression pattern using a fluorescent protein. (B) Flies heterozygous for a severe LoF allele (fry¹) and a deficiency allele (fry^Df) are lethal at early developmental stages. The fry^T2A-GAL4 allele fails to complement both fry¹ and fry^Df alleles. The stage-specific lethality rates are indicated in the table. Lethality caused by the loss of fry is rescued by the introduction of a genomic rescue (GR) construct. (C) Fry¹ homozygous mutant clones were generated in the fly wings using the FRT/FLP system. The loss of fry causes the clustered wing hair (furry) phenotype in clones. (D) Fry¹ homozygous mutant clones were generated in the fly compound eyes using the FRT/FLP system. Homozygous fry mutant clones are not present, and the eyes are small and rough, indicating that loss of fry causes cell lethality in the developing eye. Scale bar, 200 μm.

Figure 4

Fry is expressed in neurons in the fly CNS and localizes to the cytoplasm (A and B) The expression of nuclear localized mCherry (mCherry.nls) was driven by the fry^T2A-GAL4 allele (fry^T2A-GAL4> mCherry.nls). The larval CNS and adult brain of fry^T2A-GAL4> mCherry.nls animals were immunostained with neuronal (Elav, A) or glial marker (Repo, B). Maximum projections of confocal z stack images are shown. Fry is expressed in neurons (A) but not in glia (B) in the fly CNS. In the larval CNS, fry is expressed more widely and strongly in the ventral nerve cord (yellow solid square) than in the brain lobes (yellow dashed square) (A). Single-plane, high magnification images of the regions indicated by the white dashed squares are shown on the right to visualize the colocalizations between mCherry and the immunostaining signals. Colocalizations are indicated by the arrows. Scale bars, 100 μm. (C) The localization of Fry in neurons was visualized by the GFP-tagged protein expressed by the fry^GFP allele. A single-plane image of the adult brain is shown. Fry localizes to the neuropil areas and the cytoplasm in the neuron bodies. Nuclei are marked by 4',6-diamidino-2-phenylindole (DAPI) staining (magenta). High magnification images of the regions indicated by the white dashed squares are shown on the right to visualize the localization of Fryin the cell bodies. Scale bar, 100 μm.

Figure 5

The fry p.Phe2746Ser variant, but not the other assayed variants, phenocopies severe LoF variants (A) The fry p.Phe2746Ser variant (both PE and RMCE alleles) fails to complement the fry severe LoF variants, while the other three variants complement the severe LoF variants. The lethality of both fry^{p.Phe2746Ser_PE}/fry¹ and fry^{p.Phe2746Ser_RMCE}/fry¹ animals are rescued by the introduction of the genome rescue construct. (B) Fry homozygous mutant clones were generated in the fly wings using the FRT/FLP system. Both fry^{p.Phe2746Ser_PE} and fry^{p.Phe2746Ser_RMCE} alleles cause clustered wing hair phenotype in the homozygous mutant clones, phenocopying the severe LoF fry¹ allele. (C) Fry homozygous mutant clones were generated in the fly compound eyes using the FRT/FLP system. Both fry^{p.Phe2746Ser_PE} and fry^{p.Phe2746Ser_RMCE} alleles cause cell lethality in the homozygous mutant clones as well as small and rough eyes, phenocopying the severe LoF fry¹ allele. (D) The wings of fry^{p.Phe2024Leu_RMCE}/fry¹, fry^{p.Ser2910Ile_RMCE}/fry¹, and fry^{p.Tyr3410Cys_RMCE}/fry¹ flies do not exhibit clustered wing hair phenotype. (E) The compound eyes of fry^{p.Phe2024Leu_RMCE}/fry¹, fry^{p.Ser2910Ile_RMCE}/fry¹, and fry^{p.Tyr3410Cys_RMCE}/fry¹ flies do not exhibit any obvious morphological defect.

Figure 6

Fry p.Phe2024Leu and p.Tyr3410Cys variants behave as partial LoF variants (A and B) The time courses from egg laying to L2/L3 molting (A) and to puparia formation (B) were measured in animals heterozygous for a tested missense allele and fry¹ allele. Compared with the fry^p.WT_RMCE/fry¹ controls, fry^{p.Phe2024Leu_RMCE}/fry¹ and fry^{p.Tyr3410Cys_RMCE}/fry¹ animals present with developmental delays in both measurements. The delays are rescued by the introduction of a genome rescue (GR) construct. The fry^{p.Ser2910Ile_RMCE}/fry¹ animals do not show any delay in development. (C–F) ERGs were recorded in animals heterozygous for a tested missense allele and fry¹ allele (C). ON (D) and OFF (E) transients as well as amplitudes (F) were quantified. The ON and OFF transients in fry^{p.Phe2024Leu_RMCE}/fry¹ flies significantly decrease compared with the control and GR construct rescues the decreases. In contrast, fry^{p.Ser2910Ile_RMCE} and fry^{p.Tyr3410Cys_RMCE} do not cause a significant change in ERG. (A, B, and D–F) Results are presented as means ± SEM. Numbers of animals (n values) in each group are indicated under the bars. Results in (A) and (B), as well as results in (D–F), were obtained from the same samples. Statistical analyses were performed via unpaired Student’s t test. ns, not significant; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.