Loss of function of KIAA2022 causes mild to severe intellectual disability with an autism spectrum disorder and impairs neurite outgrowth

Abstract

Existence of a discrete new X-linked intellectual disability (XLID) syndrome due to KIAA2022 deficiency was questioned by disruption of KIAA2022 by an X-chromosome pericentric inversion in a XLID family we reported in 2004. Three additional families with likely pathogenic KIAA2022 mutations were discovered within the frame of systematic parallel sequencing of familial cases of XLID or in the context of routine array-CGH evaluation of sporadic intellectual deficiency (ID) cases. The c.186delC and c.3597dupA KIAA2022 truncating mutations were identified by X-chromosome exome sequencing, while array CGH discovered a 70 kb microduplication encompassing KIAA2022 exon 1 in the third family. This duplication decreased KIAA2022 mRNA level in patients' lymphocytes by 60%. Detailed clinical examination of all patients, including the two initially reported, indicated moderate-to-severe ID with autistic features, strabismus in all patients, with no specific dysmorphic features other than a round face in infancy and no structural brain abnormalities on magnetic resonance imaging (MRI). Interestingly, the patient with decreased KIAA2022 expression had only mild ID with severe language delay and repetitive behaviors falling in the range of an autism spectrum disorder (ASD). Since little is known about KIAA2022 function, we conducted morphometric studies in cultured rat hippocampal neurons. We found that siRNA-mediated KIAA2022 knockdown resulted in marked impairment in neurite outgrowth including both the dendrites and the axons, suggesting a major role for KIAA2022 in neuron development and brain function.

Figure 1.

Pedigrees of the four families are correlated with their molecular findings. (A) Family 1: cytogenetics breakpoints (taken from ref 6) of X-chromosome pericentric inversion. (B) Family 2: electropherogram of KIAA2022 frameshift c.3597dupA mutation. (C) Family 3: quantitative PCR performed on genomic DNA or cDNA for KIAA2022 and dosage of KIAA2022 using cDNA prepared with mRNA extracted from lymphoblasts of patient III,:3. For both the panels, each reaction was performed twice in triplicate. The expression level of the control samples was normalized using Gapdh or Adora2B. Histograms indicate KIAA2022 mRNA decreased production. (D) Family 4: Electropherogram of KIAA2022 indicating c.138 delC frameshift mutation. Amino-acid change is indicated in bold.

Figure 2.

Facial appearance of Patients 1–7: a round face is observed in young age. All patients have strabismus. Note grimacing of Patients III 1 and IV 6.

Figure 3.

KIAA2022 knockdown leads to suppression of neurite growth. (A) Primary cultured hippocampal neurons were prepared from embryonic day 18 rat embryos as previously described. Neurons were transfected with KIAA2022-specific siRNA (siRNA) or a scrambled control siRNA (scRNA), together with a fluorescent protein DsRED to indicate transfection. Neurons were then maintained in Neurobasal medium in a humidified incubator containing 5% CO₂. Two days after transfection, neurons were fixed, permeabilized and immunostained for KIAA2022. Whereas neurons transfected with scRNA showed normal levels of KIAA2022, a marked reduction in KIAA2022 was shown in neurons expressing siRNA. Transfected neurons were indicated by DsRED (arrows). (B) Neurons were transfected with KIAA2022-specific siRNA (siRNA) or a scrambled control siRNA (scRNA), together with enhanced green fluorescent protein (EGFP). Two days after transfection, neurons were fixed, permeabilized and immunostained for TuJ-1 (tubulin beta-III) to indicate all neuritis. Transfected neurons were indicated by EGFP (arrows). (C–F) Measurements on neurite length, including total neurites, singe neurite average, total dendrites and average length of individual dendrites. *P < 0.05, Student's t test.

Figure 4.

KIAA2022 knockdown inhibits axon development. (A) 48 h after transfection, neurons were immunostained for the axon-specific protein tau. (B) Axon growth was markedly reduced in siRNA-expressing neurons compared with the scRNA control. *P < 0.05, Student's t test.