Bi-allelic Variants in IQSEC1 Cause Intellectual Disability, Developmental Delay, and Short Stature

Abstract

We report two consanguineous families with probands that exhibit intellectual disability, developmental delay, short stature, aphasia, and hypotonia in which homozygous non-synonymous variants were identified in IQSEC1 (GenBank: NM_001134382.3). In a Pakistani family, the IQSEC1 segregating variant is c.1028C>T (p.Thr343Met), while in a Saudi Arabian family the variant is c.962G>A (p.Arg321Gln). IQSEC1-3 encode guanine nucleotide exchange factors for the small GTPase ARF6 and their loss affects a variety of actin-dependent cellular processes, including AMPA receptor trafficking at synapses. The ortholog of IQSECs in the fly is schizo and its loss affects growth cone guidance at the midline in the CNS, also an actin-dependent process. Overexpression of the reference IQSEC1 cDNA in wild-type flies is lethal, but overexpression of the two variant IQSEC1 cDNAs did not affect viability. Loss of schizo caused embryonic lethality that could be rescued to 2^nd instar larvae by moderate expression of the human reference cDNA. However, the p.Arg321Gln and p.Thr343Met variants failed to rescue embryonic lethality. These data indicate that the variants behave as loss-of-function mutations. We also show that schizo in photoreceptors is required for phototransduction. Finally, mice with a conditional Iqsec1 deletion in cortical neurons exhibited an increased density of dendritic spines with an immature morphology. The phenotypic similarity of the affecteds and the functional experiments in flies and mice indicate that IQSEC1 variants are the cause of a recessive disease with intellectual disability, developmental delay, and short stature, and that axonal guidance and dendritic projection defects as well as dendritic spine dysgenesis may underlie disease pathogenesis.

Keywords: BRAG2; Drosophila; autosomal recessive; axon guidance; dendritic spines; schizo, mice.

Figure 1

Segregation of IQSEC1 Variants in Two Families with Overlapping Neurodevelopmental Phenotypes Pedigree trees showing segregation of rare homozygous pathogenic IQSEC1 variants c.1028C>T (p.Thr343Met) in family 1 (A) and c.962G>A (p.Arg321Gln) in family 2 (B). Dashed line indicates the relationship by history; exact relationship is not clear (B). Unfilled shapes denote healthy while shaded shapes denote affected individuals; squares are males; circles are females; small triangle represent stillborn and double horizontal lines denote consanguineous marriages.

Figure 2

Schizo Is a Functional Homolog of IQSEC1 in Fly (A) Schizo and IQSEC1 are evolutionarily conserved and share IQ-like motif, Sec7, and PH domains. (B) Overexpression of IQSEC1 at 25°C is toxic, but the human IQSEC1 variants are not toxic when expressed under the same conditions. (C) Structure of schizo and its transcripts. MiMIC transposable element insertions in schizo are embryonic lethal. The MiMIC siz^MI01727 was converted using RMCE and an artificial exon that encodes GFP was introduced (siz^{MI01727-GFSTF.2}) to determine protein localization. (D) Embryonic lethality of siz^M13078 and siz^MI01727 are partially rescued by human IQSEC1 WT but not by IQSEC1 p.Thr343Met or IQSEC1 p.Arg321Gln. 0% of eggs of homozygous mutants (siz^MI13078 and siz^MI01727, n > 500) hatched. At room temperature, 70% (n = 120) of the eggs hatched when the UAS-IQSEC1 reference was ubiquitously overexpressed (siz^MI01727/siz^MI01727;act-Gal4/UAS-IQSEC1 ref) but 80% (n = 84) of hatched larvae died as 2^nd instar larvae (∼20% of hatched larvae die in 1^st instar larvae stage). None of embryos (n = 150) hatched when the UAS-IQSEC1 variants were ubiquitously overexpressed (siz^MI01727/siz^MI01727;act Gal4/UAS-IQSEC1 [p.Thr343Met or p.Arg321Gln]).

Figure 3

Schizo Is Localized in Glia and Neurons in Fly (A) siz^{MI01727-GFSTF.2} (siz-GFP) embryos were stained with anti-Repo (glia) and anti-Elav (neurons) antibodies. Siz-GFP is broadly localized in the nervous system in fly embryos, including neurons and glia. The scale bar is 50 μm. (B) In adult heads, Siz-GFP (green) is localized in the neuropil and cell bodies of neurons and glia. Elav staining labels the nuclei of the neurons (magenta). The scale bar represents 50 μm. (C) Analysis of lysates from siz-GFP flies revealed three different kinds of transcripts.

Figure 4

Schizo Is Required for Proper Axon Guidance in CNS and PNS (A) Homozygous siz^MI01727 embryos show commissural defects (white arrows). (B) Homozygous siz^MI01727 embryos exhibit severe axonal guidance defects in the peripheral neurons of all segments (yellow arrows). The scale bar represents 50 μm.

Figure 5

Schizo Is Required in the Fly Visual System Lowering the level of Schizo in neurons (elav > siz RNAi-1 and elav > siz RNAi-2) caused dramatically reduced amplitudes and loss of on- and off-transients of ERGs when compared to control (elav > Luciferase RNAi). Statistical analyses are one-way ANOVA followed by a Tukey post hoc test. Mean ± SEM; ^∗∗∗∗p < 0.001.

Figure 6

IQSEC1 Depletion in Mouse Cortex Results in Dendritic Spines of Immature Morphology (A) Western blot revealing IQSEC1 in the cerebral cortex of wild-type (WT), NEX-Cre-negative (ctrl), and NEX-Cre-positive (ΔIQSEC1) Iqsec1^fl/fl mice. (B) Representative images of dendritic segments of cortical layer V neurons of thy1-GFP-positive ctrl and ΔIQSEC1 mice. Scale bar: 5 μm. (C) Spine density was increased upon IQSEC1 depletion. n = 39–47 dendrites of four mice per genotype, ^∗p < 0.0001. (D) Spine body length was not changed upon IQSEC1 depletion. n = 39–47 dendrites, p = 0.868. (E) Spine head diameter was decreased upon IQSEC1 depletion. n = 39–47 dendrites, ^∗p < 0.0001. Data in (C)–(E) are represented as total range (whiskers), interquartile range (box), and median (line inside box). (F and G) Density of spines with small heads (spine head diameter < 0.6 μm) was selectively increased upon IQSEC1 depletion. Shown are the densities of spines grouped into body length bins of 0.2 μm (F) or grouped into head diameter bins of 0.1 μm (G). Values on x-axes indicate the maximum of each bin. (H) Cumulative frequency distribution of spine maturity index (0.0–1.5), calculated as the ratio of head diameter (d_h) to body length (l_b) of each individual spine of the analyzed population (ctrl, n = 1,812; ΔIQSEC1, n = 1,821), shifted to the left upon IQSEC1 depletion.