Loss-of-function variants in TIAM1 are associated with developmental delay, intellectual disability, and seizures

Abstract

TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. Here, we describe five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. We found that the Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects, are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. We also assessed the function associated with three TIAM1 variants carried by two of the probands and compared them to the TIAM1 Ref cDNA function in vivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, we provide evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans.

Keywords: Drosophila; Sif; TIAM1; developmental delay; intellectual disability; seizures; speech delay; still life.

Graphical abstract

Figure 1

Brain MRI images of proband 2 (A–D) Brain MRI for proband 2 was obtained to document the acquired macrocephaly (Z ≥ 2) at 14 months of age. 3T coronal T1 images (A and B) show prominence of the extra-axial fluid spaces involving both hemispheres, left greater than right, with prominence of the cortical sulci for age, enlargement of the lateral and third ventricles, and cavum septum pellucidum and cavum vergae. The brain parenchyma items show preservation of the gray-white matter differentiation with a myelination pattern appropriate for age. Axial T2 (C) and T2 FLAIR (D) images demonstrate intact and symmetric subcortical structures without evidence of heterotopia or other migrational defects. Also visible are prominent subarachnoid spaces with superimposed subdural collections most consistent on susceptibility-weighted imaging (not shown) as chronic subdural hemorrhage.

Figure 2

sif is the TIAM1 ortholog in fly (A) Prediction of sif human homologs using DIOPT (DRSC integrative ortholog prediction tool). (B) Protein domain structure of TIAM1 and Sif. (C) Genomic structure of sif locus and reagents used in this study. For detailed information on these reagents, please see Tables S1 and S2. (D) Schematic of sif^T2A-GAL4 generation by RMCE using sif^MiMIC-1 (sif^MI02376, BDSC #35834). SA, splice acceptor; P, attP; B, attB; y+, yellow+ gene as a marker. (E) Relative sif mRNA expression levels are lower than 20% in sif^T2A-GAL4 mutant larvae when compared to controls (w¹¹¹⁸) based on real-time PCR using primers shown in (C). “sif-all” targets all of the 13 sif transcripts, while “sif-1” targets 8 of the 13 transcripts (sif-RA, RB, RC, RI, RL, RM, RN, and RO). Each sample contains 3–5 L3 larvae. Data are represented as mean ± SEM. Unpaired t tests. ∗p < 0.05.

Figure 3

sif is expressed primarily in neurons of the fly CNS (A–D) The L3 larval CNS (A, C) and adult brains (B, D) of sif^T2A-GAL4 allele-driven expression of UAS-mCherry.NLS (nuclear mCherry) were stained with markers for neurons (Elav, A and B) or glia (Repo, C and D). Single-slice confocal images are shown. (A′–D′) Images are from dashed squares of indicated regions to visualize the cellular co-localizations with Elav (A′ and B′) or Repo (C′ and D′). Scale bars: 100 μm. Note that sif is expressed in neurons. (E and F) The sif^T2A-GAL4 allele-driven expression of UAS-mCD8::RFP (membrane-bound RFP) shows that sif is broadly detected in the CNS of L3 larvae (E) and adult flies (F). Scale bars: 100 μm.

Figure 4

Loss of sif causes semi-lethality, and the surviving flies exhibit climbing defects and seizure-like behaviors (A) Table summarizing complementation tests of sif LoF mutants. sif LoF mutants are semi-lethal and have severe (“ǂ”) or mild (“#”) climbing defects. The lethality of “sif^ES11” and “sif^MiMIC-2” homozygotes is likely caused by second-site mutations. (B) sif LoF mutants have severe climbing defects 3–5 days after eclosion. Flies were raised at 22°C. (C) sif LoF mutants have a bang-sensitive phenotype, which can be rescued by UAS-sif expression. Flies were raised at 22°C and tested at day 3–5. (D and E) sif LoF mutants have heat-induced seizure-like behaviors. Flies in vials were immersed in a 42°C water bath for 30 s. The percentage of flies that are unable to keep an upright position in sif LoF mutant group is significantly higher than the control groups (D), and the mutants could not recover 20 s after heat shock (E). The seizure-like behaviors can be rescued by UAS-sif expression. Flies were raised at 22°C and tested at day 3–5. Data are represented as mean ± SEM. Unpaired t tests. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; n.s., no significance.

Figure 5

Neurological phenotypes of sif LoF mutants are mainly caused by neuronal loss of sif (A) Real-time PCR analysis of sif mRNA levels in ubiquitous sif knockdown flies. Each sample contains 3–5 L3 larvae. sif-RNAi-1 causes a higher knockdown efficiency than sif-RNAi-2. (B) Survival rates of ubiquitous expression of sif RNAi at 25°C. sif knockdown with RNAi-1 causes semi-lethality, while RNAi-2 knockdown does not affect viability, indicating a strong dose dependency. (C) Ubiquitous sif knockdown causes climbing defects in a dose-dependent manner. Flies were tested at day 3. (D and E) Ubiquitous sif knockdown causes heat-induced seizure-like behaviors in a dose-dependent manner. The percentage of flies that are unable to keep an upright position in the sif RNAi-1 group is significantly higher than controls (D), and the majority of flies are not able to recover 20 s after heat shock (E). Similar but milder phenotypes were seen in the sif RNAi-2 group. Flies were tested at day 3. (F) Neuronal sif knockdown causes climbing defects. Flies were allowed to climb for 5 s. (G and H) Neuronal sif knockdown causes heat-induced seizures. The percentage of flies that are unable to keep an upright position in the sif RNAi-1 group is significantly higher than controls (G), and the majority of flies in the sif RNAi-1 group did not recover in 20 s after heat shock (H). Flies were tested at day 5. (I) Glial sif knockdown does not cause climbing defects. Flies were tested at day 15. All the animals were raised at 25°C unless otherwise mentioned. Data are represented as mean ± SEM. Unpaired t tests. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001; n.s., no significance.

Figure 6

sif LoF rescue studies show that TIAM1 Ref has partial rescue ability, but the variants have variable rescue abilities (A) The rescue ability of UAS-TIAM1 Ref decreases with increased temperature. UAS-lacZ was used as a negative control that doesn’t alter the survival rate. Fly UAS-sif was used as a positive control, and it completely rescues the survival rate of sif LoF mutants. (B) The semi-lethal phenotype of sif^T2A-GAL4 mutants can be fully rescued by expression of fly sif cDNA and partially rescued by TIAM1 Ref cDNA. The TIAM1 variant p.Arg23Cys shows reduced rescue ability, while p.Leu862Phe and p.Gly328Val show similar rescue abilities when compared to TIAM1 Ref. Similar but lower rescue abilities were seen in sif^T2A-GAL4 homozygous background, suggesting two GAL4 copies cause higher TIAM1-induced toxicity. Flies were raised at 18°C. (C) sif LoF mutants have significantly shorter lifespans, which can be rescued by UAS-sif and partially rescued by UAS-TIAM1 Ref, while p.Arg23Cys shows reduced rescue ability. Flies were raised and kept at 18°C. Data are represented as mean ± SEM. Unpaired t tests (B) and log-rank (Mantel-Cox) and Gehan-Breslow-Wilcoxon tests (C). ∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; n.s., no significance.

Figure 7

Ectopic expression of WT fly sif or human TIAM1 Ref is toxic, but human TIAM1 variants are less toxic (A) Table summarizing the lethality phenotypes of ubiquitous overexpression (da-GAL4) of sif, TIAM1 Ref, and variants. (B) Quantification of survival stages of ubiquitous overexpression of sif, TIAM1 Ref, and variants at 22°C. Note that the variants are less toxic. Data are represented as mean ± SEM. (C) Representative pictures showing that wing-specific overexpression (nub-GAL4) of sif and TIAM1 Ref causes similar vein loss and blistery wing phenotypes. The defects are highlighted in red dashed circles. Flies were raised and kept at 25°C.