Variants in the degron of AFF3 are associated with intellectual disability, mesomelic dysplasia, horseshoe kidney, and epileptic encephalopathy

Abstract

The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.

Keywords: AFF3; AFF4; horseshoe kidney; intellectual disability; mesomelic dysplasia.

Figure 1

AFF3 and AFF4 degron motif variants (A) Schematic protein structure of ALF proteins from the amino terminus: an N-terminal homology domain (NHD), the AF4/LAF4/FMR2 (ALF) homology domain containing the SIAH-binding degron motif, a serine-rich transactivation domain (TAD), a bipartite nuclear/nucleolar localization sequence (NLS), and a C-terminal homology domain (CHD). The sequences of the degron motifs of AFF3 and AFF4 are shown above. The residues modified in the KINSSHIP probands described in this manuscript and individuals affected by CHOPS^, are highlighted in bold and numbered. The extent of the 496 kb deletion identified in this work and the extent of the 500 kb deletion previously described are indicated by black bars. A red arrow pinpoints the position of the degron motif. (B) Amino acid sequence alignment of human AFF1, AFF2, AFF3, and AFF4 proteins (ENSP00000305689, ENSP00000359489, ENSP00000317421, and ENSP00000265343, respectively) showing the highly conserved degron motif (red rectangle) of the ALF homology domain that provides the binding moiety to the SIAH ubiquitin-ligase. Sequence alignment was performed with Clustal Omega and edited with Jalview. Shading is proportional to conservation among sequences. (C) Amino acid sequence alignment of different AFF3 vertebrate orthologs showing the conservation of the degron motif (red rectangle). Accession numbers are ENSP00000317421 (human), ENSMUSP00000092637 (mouse), ENSFCAP00000024603 (cat), ENSLAFP00000010776 (elephant), ENSPSIP00000007060 (chinese turtle), ENSACAP00000008035 (anole lizard), and ENSPMAP00000008605 (lamprey). (D) 3D modeling of the binding of human AFF3 degron to the mouse Siah ubiquitin ligase. We downloaded PDB: 2AN6, in Swiss-PdbViewer and used it as a template to align the human SIAH ubiquitin ligase (UniProt: Q8IUQ4). With respect to the mouse crystal structure, the only difference is the presence of an aspartic acid residue instead of a glutamic acid at position 116. We then aligned the region of AFF3 containing the degron motif (LRPVAMVRPTV) onto the Siah-interacting protein peptide present in the crystal structure (QKPTAYVRPMD) to highlight the position of the variants reported in this study. For clarity, only sidechains of the core degron motif (Pro256, Ala258, Val260, and Pro262) are shown. The sidechains of KINSSHIP mutated residues are highlighted in yellow. The core degron motif adopts a beta-strand conformation directly contacting the ubiquitin ligase-binding groove. The sidechains of Ala258 and Val260 are embedded into binding pockets too small to accommodate larger sidechains. They are in direct proximity of Siah residues Thr156 (pink) and Met180 (cyan), identified as key binding residues in a series of pull-down assays. Replacing Pro256 with Leucine, a residue with a longer sidechain that will be positioned in proximity of Siah residue Leu158 (orange), could affect the backbone kink normally conferred by the conserved Proline. The longer sidechains of p.Pro256Leu, p.Ala258Thr, p.Ala258Ser, and p.Ala258Val variants and the smaller p.Pro256Ala and p.Val260Gly are likely to weaken or prevent the interaction with the ligase.

Figure 2

AFF3 stability and evolution (A) Immunoassays comparing the stability of wild-type and mutated forms of AFF3 proteins. We transiently co-transfected HEK293T cells with expression vectors encoding FLAG-tagged AFF3^wild-type (WT), AFF3^A258T (A258T), or AFF3^V260G (V260G) proteins and HA-tagged SIAH1 E3-ligase (HA-SIAH1) or an empty vector (empty) in presence/absence of increasing amount of the MG132 proteasome inhibitor (0, 2, and 10 μM). Protein extracts were separated by capillarity on a Jess system and immunoassayed with an anti-FLAG antibody (upper portion) and an anti-HA antibody (bottom portion). The image shows a typical example of eight replicas performed in the same conditions. Loading control and normalization are shown in Figure S1. (B) ALF protein phylogeny. The maximum likelihood phylogenetic tree was constructed with 26 AFF amino acid sequences: mammals: Homo sapiens AFF1 (NP_001160165.1), AFF2 (NP_002016.2), AFF3 (NP_002276.2), and AFF4 (NP_055238.1); birds: Gallus gallus AFF1 (XP_004941155.1), AFF2 (XP_015134139.2), AFF3 (XP_015133277.1), and AFF4 (XP_015149549.1); reptiles: Anolis carolinensis AFF1 (XP_008109400.2), AFF2 (XP_016851830.1), AFF3 (XP_008118477.1), and AFF4 (XP_003217431.2); amphibians: Xenopus laevis AFF1 (XP_018108715.1), AFF2 (XP_018088502.1), AFF3 (XP_018104097.1), and AFF4 (XP_018107624.1); bony fishes: Danio rerio AFF2 (XP_002664429.2), AFF3 (XP_021334573.1), and AFF4 (XP_005173956.1); cartilaginous fishes: Callorhinchus milii AFF1 (XP_007895125.1), AFF2 (XP_007891068.1), AFF3 (XP_007884050.1), and AFF4 (XP_007889648.1); lamprey: Petromyzon marinus AFF (PMZ_0026877); tunicate: Ciona intestinalis AFF (XP_018673247.1); and invertebrates: Drosophila melanogaster AFF (NP_722863.1). The bootstrap consensus tree inferred from 100 replicates is shown.

Figure 3

Photographs of KINSSHIP-affected individuals with AFF3 de novo missense variants Proband 4 at 2 years 6 months old (A). Proband 7 at 18 years old (B and I). Proband 8 at 9 months (C) and 21 years old (D and J). Proband 9 at 1 year 7 months (E) and 16 days old (N and O). Proband 10 at 9 years old (F and K–M). Proband 11 at 8 years old (G). Proband 12 at 7 years 9 months old (H and P–R). Proband 15 at 11 years old (S). Note the synophrys and micrognathia, protruding ears, large nose with prominent nasal tip, and prominent teeth in probands 7 (B), 8 (D), 10 (F), and 12 (H), as well as hypertrichosis of the limbs (I, J, M, and P). Together with probands 9 and 11, they exhibit thick hair, long eyelashes, and a wide mouth (E and G). Facial features coarsen with age as shown by pictures of proband 8 at different ages (C and D), explaining the more delicate features of younger probands (A and E). AFF3 de novo missense variant carriers also have hypoplastic talipes and abnormalities of toes (I, J, and M–S). Proband 10 also shows clinodactyly and soft tissue syndactyly of both hands (K and L).

Figure 4

X-rays of KINSSHIP-affected individuals with de novo missense variants in AFF3 (A–D) Proband 7 at 18 years old. Severe scoliosis (A), dorsal and radial bowing of the radius and "V-shaped" proximal carpal bones as seen in Madelung deformity (B), metaphyseal widening and hypoplastic fibula (C), and hypoplastic talipes (D). (E–I) Proband 8 at 21 years old. Static scoliosis (E) and short ulna and radius (F). Note erratic articulation of the styloid process of the ulna on the radius rather than on the carpal bones. Congenital fusion of the bases of the second and third right metatarsals (G), forearm with dislocation of proximal radius (H), and hypoplastic and short bowed tibias with enlarged metaphyses (I). (J–L) Proband 9 at 10 months old. Right foot with 4^th and 5^th metatarsals synostosis (J) and left foot missing the lateral ray (K) and extremely short rectangular fibula and bowed tibia (L). (M) Proband 11 at 8 years old. Hypoplastic fibula (M). (N–P) Proband 15 at 10 years old. Scoliosis and cervical ribs (N), bowed radius with proximal dislocated head (O), and distal shortening of ulna bowed tibia, severely hypoplastic fibula and oligodactyly (P).

Figure 5

Neuroanatomical defects in Aff3^−/− mice (A–F) Merged double-stained sections in Aff3^−/− mice (right of dashed lines) and their matched controls (WT, wild-type, left of dashed lines) at the striatum (A) and at the hippocampus (B) levels with schematic representation of the affected areas (C and D). Histograms showing the percentage of increase or decrease of parameters in measured areas as compared to the controls for striatum (E) and hippocampus (F) sections. Red shading is proportional to the stringency of the significance threshold. Numbers indicate studied areas: 1, total brain area; 2, lateral ventricles; 3, cingulate cortex (section 1) and retrosplenial cortex (section 2); 4, corpus callosum; 5, caudate putamen (section 1) and hippocampus (section 2); 6, anterior commissure (section 1) and amygdala (section 2); 7, piriform cortex; 8, motor cortex; 9, somatosensory cortex; 10, mammillo-thalamic tract; 11, internal capsule; 12, optic tract; 13, fimbria; 14, habenular; 15, hypothalamus; 16, third ventricle. Results demonstrate an enlargement of lateral ventricles (LVs; p = 1.24E−4 on section 1, p = 4.64E−2 on section 2) and a smaller genu of the corpus callosum (gcc; decreased corpus callosum size p = 6.35E−2 indicated by the black dash and double arrow, decreased bottom width of the corpus callosum p = 3.02E−6 and decreased height of the corpus callosum p = 4.96E−2). Other phenotypes such as atrophy of the anterior commissure (aca; p = 1.02E−2) and smaller hippocampus (p = 4.02E−2) are significant if using a less stringent cutoff.

Figure 6

Animal models (A) Schematic representation of the deletion generated in mice ES cells with the CRISPR/Cas9 system, which models the mutation observed in the historical deletion proband.^, (B) Skeletal staining of E18.5 mouse embryos shows mesomelic dysplasia with triangular tibia and hypoplastic fibula (see Figure 3 in Kraft et al.³⁸), as well as a hypoplastic pelvis in Aff3^del/del mice, especially noticeable in the iliac wing (black arrows) and acetabulum (orange arrows); perturbations also observed in the historical deletion proband. (C) Delayed ossification of flat bones in the skull of Aff3^del/del mice. (D) Lateral (top line) and dorsal (bottom line) views of the observed phenotypes of 4 dpf AB-WT zebrafish embryos injected with human AFF3 mRNA (hAFF3). hAFF3-injected zebrafish embryos exhibit severe developmental defects, including a bent body axis and yolk sac edema (D3–D6), as well as extreme malformations with absence of body axis, tail, and fins and cyclopia (D7 and D8). Embryos with normal development are displayed for comparison (D1 and D2). (E) Proportions of normal and developmentally defective 4 dpf AB-WT zebrafish embryos upon injection of increasing doses of hAFF3 (left panel) and hAFF4 (right panel) mRNA. Dark and light colors indicate developmentally defective and normal animals, respectively. Control injections with phenol red show no significant (ns) differences with WT in both AFF3 and AFF4 experiments (Fisher’s exact test, p = 0.09 and p = 0.12, respectively). hAFF3 mRNA injection significantly increases the number of zebrafish with developmental defects when compared with controls starting from 150 ng (^∗; p = 0.03) and reinforced at 300 ng (^∗∗∗; p = 3.2E−5). AFF4 injections do not have a significant impact on zebrafish development compared to WT, even at the same dose (300 ng, p = 0.29).