Exonic deletions in AUTS2 cause a syndromic form of intellectual disability and suggest a critical role for the C terminus

Abstract

Genomic rearrangements involving AUTS2 (7q11.22) are associated with autism and intellectual disability (ID), although evidence for causality is limited. By combining the results of diagnostic testing of 49,684 individuals, we identified 24 microdeletions that affect at least one exon of AUTS2, as well as one translocation and one inversion each with a breakpoint within the AUTS2 locus. Comparison of 17 well-characterized individuals enabled identification of a variable syndromic phenotype including ID, autism, short stature, microcephaly, cerebral palsy, and facial dysmorphisms. The dysmorphic features were more pronounced in persons with 3'AUTS2 deletions. This part of the gene is shown to encode a C-terminal isoform (with an alternative transcription start site) expressed in the human brain. Consistent with our genetic data, suppression of auts2 in zebrafish embryos caused microcephaly that could be rescued by either the full-length or the C-terminal isoform of AUTS2. Our observations demonstrate a causal role of AUTS2 in neurocognitive disorders, establish a hitherto unappreciated syndromic phenotype at this locus, and show how transcriptional complexity can underpin human pathology. The zebrafish model provides a valuable tool for investigating the etiology of AUTS2 syndrome and facilitating gene-function analysis in the future.

Figure 1

Overview of AUTS2 Aberrations in the Probands The location of the deletions is indicated by the red bars, the inversion breakpoint is indicated by an arrowhead, and the translocation breakpoint area is indicated by the red horizontal bracket. CNVs extracted from the Database of Genomic Variants are in purple (CNVs found in bacterial-artificial-chromosome studies are not included). The AUTS2 syndrome severity score of the probands is shown on the right. Darker shades indicate a more severe and/or more specific phenotype. Color coding of AUTS2 syndrome severity scores is as follows: white, <7; light gray, 7–12; gray, 13–18; and dark gray, >18. See also Figure S2 and Table S7.

Figure 2

Facial Characteristics of Cases with an AUTS2 Aberration (A) Case 1 at age 3 years shows no dysmorphic features. (B and L) Front (B) and side (L) views of case 4 at age 2.5 years show a repaired cleft lip, mild proptosis, and short and mild upslanting palpebral fissures. (C) The mother of case 4 shows a repaired cleft lip, ptosis, and retrognathia. (D) Case 5 at age 3 years shows highly arched eyebrows, mild downslanting palpebral fissures, epicanthal folds, and a short philtrum. (E, F, M, and N) Front (E) and side (M) views of case 6 at age 6 years. She is hyperteloric and has ptosis and downslanting palpebral fissures, a short philtrum, and a narrow mouth similarly to her brother, shown in (F) and (N) at the age of 10 years. (G and O) Front (G) and side (O) views of case 9 at age 32 years show hypertelorism, proptosis, upslanting palpebral fissures, a short upturned philtrum, and a narrow mouth. (H) Case 10 at age 2 years shows a prominent nasal tip, anteverted nares, and a short philtrum. (I and P) Front (I) and side (P) views of case 13 at age 5.5 years show hypertelorism, ptosis, a broad nasal bridge, a short and upturned philtrum, and a narrow mouth. (J, K, and Q) Case 15 at age 1 year (J) and 4.8 years (K and Q) shows a broad nasal bridge, short palpebral fissures and a short philtrum, and a narrow mouth. See also Table 1 and Table S7.

Figure 3

Scatter Plot of the AUTS2 Syndrome Severity Score for Disruptions Affecting the N or C Terminus of AUTS2 Scatter plot of the AUTS2 syndrome severity score for disruptions that affect the highly conserved amino acid sequence block encoded by exons 9–19 (yes) and the deletions not affecting this amino acid sequence (no) (see also Table S7 and Figure 4). The numbers refer to case numbers. The following abbreviations are used: f, father of patient x; m, mother of patient x; and s, sibling of patient x (see Table S7). The AUTS2 syndrome severity scores between these groups of cases differ significantly (p = 0.001, Kolmogorov-Smirnov Z test).

Figure 4

Exon Organization of AUTS2 and Its Zebrafish Ortholog and Identification of a Novel Transcriptional Start Site in Exon 9 of Full-Length Human AUTS2 (A) Exon organization of AUTS2 orthologs in humans and zebrafish. Arrows indicate two TSSs used in human brain mRNA. The alternative novel TSS is located 1.17 Mb downstream of the standard TSS in the cluster containing exons 7–19. Exons 1–6 (zebrafish) represent conserved sequences that are not annotated in the current zebrafish genome (for details, see Figure S4). (B) Identification of an alternative AUTS2 transcript detected in human brain mRNA by 5′ RACE. The alternative transcript starts in the center of exon 9 (asterisk) and contains the indicated cDNA sequence (in italics). The mRNA was spliced to exon 10 with the second of two known splice donor sites in exon 9, resulting in the incorporation of seven alternatively spliced amino acids (rectangle). The alternative mRNA uses the same reading frame as the conventional transcript. Conventional exons are in uppercase, and introns are in lowercase. See also Figures S1 and S3.

Figure 5

Suppression of auts2 in Zebrafish Leads to Small Head Size and Craniofacial Defects (A) Lateral views of representative control embryos and embryos injected with auts2 MOs. (B) Quantification of microcephaly was performed in embryo batches injected with 4.5 ng 5′ MO (targeting exon 2 donor splice) or 6 ng 3′ MO (targeting exon 10 donor splice) plus 100 pg wild-type human AUTS2 full-length (FL) or short isoform (3′) mRNAs (n = 56–91 embryos per injection). p values are denoted on the bar graph. The following abbreviation is used: NS, nonsignificant. (C) No significant difference in body length was observed in auts2 morphants and rescued embryos at 2 dpf. Bars represent the average length of 30 embryos, which were scored blindly to injection cocktail. Data are shown as the mean ± SD. (D) Ventral views of representative control embryos and those injected with auts2 MOs (either a 3′ or 5′ MO) at 5 dpf. Cartilage structures were visualized by whole-mount Alcian-blue staining at 5 dpf, allowing measurement of the distance between ceratohyal and Meckel’s cartilages (red lines). (E) Averaged distance measurements are presented as the mean ± SEM. The corresponding p values are denoted on the bar graph (two-tailed t test comparisons). The following abbreviations are used: ch, ceratohyal cartilage; and Mk, Meckel’s cartilage.

Figure 6

Suppression of auts2 Leads to Reduced HuC/D Protein Levels and Fewer Proliferating Cells (A) Suppression of auts2 leads to a decrease of HuC/D levels at 2 dpf. Representative photographs (with HuC/D-antibody staining) show the ventral views of a control, an embryo injected with an auts2 MO, and a rescued embryo injected with an auts2 MO plus 3′ human AUTS2 mRNA at 2 dpf. HuC/D levels in the anterior forebrain of the embryo injected with the auts2 MO are considerably lower than those of the control embryo. This defect was rescued significantly by coinjection of full-length (FL) or short isoform (3′) human AUTS2 mRNAs. (B) Percentage of embryos with normal, bilateral HuC/D protein levels in the anterior forebrain (blue) or decreased and/or unilateral HuC/D protein levels (red) in embryo batches injected with auts2 MOs alone or MOs plus human AUTS2 FL or 3′ mRNA (MO + 3′ mRNA). p values are denoted on the bar graph. (C) Phosphohistone-H3 staining for proliferating cells in the zebrafish brain at 2 dpf. (D) Quantification of phosphohistone-H3-staining intensities from 20 embryos each (control embryos or embryos injected with MOs alone or MOs plus 3′ or 5′ human AUTS2 mRNA). Data are represented as the mean ± SEM. The corresponding p values are denoted on the bar graph (two-tailed t test comparisons between MO-injected and rescued embryos).