In this study, we include 23 novel and 11 previously published individuals [14,15,16], from which 30 were unrelated, diagnosed with heterozygous variants in CUX1. The cohort compromises of nine females and 25 males with a median age of 7 years (ranging from 7 months to 78 years). Table 1 summarises the clinical symptoms and Table S1 provides detailed clinical descriptions.
Phenotypic spectrum of the affected individuals—Intellectual and social development
All but one individual presented with delayed speech development (31/32, 97%). Individuals 4 and 21 initially presented with a disorder of motor development. Amongst individuals 6 years or older, formal IQ testing was available in ten Individuals. Suitable tests were provided for two individuals. If no formal test was available, the attending physician assessed the severity in an age-appropriate manner. ID was stratified as borderline (4/27, 15%), mild (8/27, 30%), moderate (7/27, 26%) and severe (2/27, 7%). Six individuals had an IQ in the normal range (6/27, 22%). Most individuals showed delayed motor development (24/32, 75%). The median age for walking was 20 months, and the medianfor first speech was 22.5 months. Six individuals were diagnosed with ASD (6/26, 23%).
We had previously noted that three individuals with a CUX1 null variant caught up on developmental milestones [14]. Now, including the individuals presented in this study, developmental catch-up was observed in seven individuals (7/18, 39% from which this information was assessable; total 21%). Specifically, individual 6 presented speech and motor delay with first words occurring within 12–42 months and walking at 36 months of life. When he enrolled in a mainstream school, speech was comparable to children of the same age (developmental catch-up between 3 and 5 years of age). He received support throughout his school years in concordance with his lower IQ. At the age of 21 years, he had normal speech, borderline ID and was diagnosed with ASD. At age 36 months, individual 25 presented with significantly delayed speech (spoke 5–10 words) and motor development, muscular hypotonia, and macrocephaly. He could speak in 2–3 word sentences at 42 months, with further speech improvements at 4.5 years of age. At 5 years of age, the speech was comparable to children of the same age. However, due to muscular hypotonia motor development was still markedly delayed. At the age of seven, the individual enrolled in a mainstream school, albeit with integration aids for motor difficulties. Formal testing revealed an IQ of 90. At age nine, he was able to participate fully in school sports. The other six individuals presented variable delay of speech development in their earlier examinations, reporting first words within 12–42 months of age. However, they had normal speech or IQ at their last exams, performed at ages ranging from 8 to 55 years. These individuals were cognitively less affected (five individuals with normal IQ and three with borderline cognition) compared to individuals without catch-up development (two individuals with normal IQ, one with borderline, eight with mild, seven with moderate, and two with severe intellectual disability).
Abnormalities of the nervous system
Muscular hypotonia was the most common neurological finding in the present cohort (13/31, 42%; median age of affected individuals: 5.3 years, ranged from 2 to 19 years). Seven individuals had mild cerebellar symptoms, including ataxia (7/23, 30%; median age of affected individuals: 6.6 years, ranged from 0.6 to 78 years), and eight individuals (8/32, 25%) developed seizures. The mean onset of seizures was 3 years and ranged between 1 and 6 years of age, with variable seizure types (including tonic-clonic and myoclonic seizures). All but two individuals became seizure-free (between 17 months and 22 years of age). Unfortunately, no further details regarding epilepsy type or EEG are available for the affected individuals. We could not observe a correlation between seizures and the severity of ID. Eight of the 20 individuals with available brain MRI imaging (performed and interpreted by each center) had no abnormalities. In the other 12 individuals, non-recurrent changes, such as a slightly prominent fourth ventricle, Chiari malformation, and white matter T2 hyperintensities, were observed (12/20, 60%; total 35%).
Additional symptoms
Six individuals exhibited short stature (6/28, 21%), and eight showed joint laxity (8/20, 40%; total 23%). Abnormalities of the cardiovascular system, including persistent ductus arteriosus, atrial septal defect, and ventricular septal defect, were observed in eleven individuals (11/26, 42%; total 32%). Additional findings include mild scoliosis (3/21, 14%; total 9%) and genital malformations such as hypospadia, micropenis, and bilateral testicular ectopia in ten males (10/16, 63%; total 40%). The examined individuals had no apparent shared facial gestalt (Fig. S1). However, ten individuals displayed abnormalities in calvarial morphology, including macrocephaly, brachycephaly, plagiocephaly, and dolichocephaly. Eight individuals had a broad forehead (8/26, 31%), and four displayed frontal bossing (4/26, 15%). Seven individuals had low-set ears (7/25, 28%), and three had retrognathia (3/21, 14%). The physican’s examination was also consistent with the facial analysis performed by GestaltMatcher [24]. Although only nine individuals consented for a facial analysis, they might share a similar facial phenotype on the cohort level, as 78% of the distribution is below the threshold (Fig. S2). In the pairwise analysis (Fig. S3), it was clear that individuals 8, 9, 14, 18, and 19 formed one cluster, and individuals 6, 7, 25, and 28 were not in the cluster. The results suggested that CUX1 individuals might share a certain degree of similarity, but some individuals presented heterogenous facial phenotypes.
We also identified two individuals with a de novo missense variant in CUX1 that affects only the transcript encoding CASP but not CUX1 (Table S1, Fig. 1). The first individual (CASP_1:c.1820T > C, p.(Met607Thr)), had congenital glaucoma and short stature but no neurological symptoms. In contrast, a second individual (CASP_2:c.1570C > T, p.(Arg524Cys)) had severe global developmental delay, hypotonia, and seizures. It is yet unclear whether these variants are causative and whether variants in CASP are associated with another distinct disorder. Therefore, we did not include these individuals in the phenotypic description of the present cohort. In addition, we gathered information on two neonates with a de novo heterozygous null variant in CUX1. However, for the phenotypic characterization, we included only postnatal individuals.
Location of missense and null variants in CUX1 with respect to the domain structure of CUX1 (GenBank: NM_001202543.2) and CASP (GenBank: NM_001913.4). Variants reported in this cohort are labeled with the corresponding p- or c-code and are indicated by a red circle (missense) or a yellow square (null variant). Variants that affect only CASP are labeled in gray, as the relevance of those variants is uncertain. Confirmed de novo variants are indicated in bold. Lines above the protein scheme indicate null variants in gnomAD with allele count (1 is not shown). Gross deletions and structural variants are indicated as bars below the protein scheme. Abbreviations: CUT: CUT domain, HD: CUT homeobox.
CUX1 genotypic spectrum
In 30 individuals, we identified a heterozygous CUX1 null variant: two splice-site variants, three gross deletions, one inversion, one translocation, eight nonsense, and 15 frameshift variants. Four individuals harbor heterozygous missense variants (Fig. 1). All variants were absent in gnomAD, except the variant c.2398del. Of the 29 individuals for whom we were able to conduct a segregation analysis, 22 had de novo variants, while seven inherited the variant from a milder affected parent (Table S1). All but four truncating variants will likely result in nonsense-mediated mRNA decay (NMD). The variant c.61C > T will likely escape NMD as it creates a stop codon within the first protein-coding 100 nucleotides [28]. As the next in-frame AUG codon is 195, possibly a shortened abnormal CUX1 will be translated. The variants c.3819delG causes a frameshift that affects the CUT homeobox, truncates the C-terminal end of CUX1, and leads to escape of NMD, as confirmed by mRNA analysis (Fig. S4). The variants c.4321dup and c.4440_4447del (individuals 28 and 29, respectively) cause a frameshift predicted to result in CUX1 elongation with an abnormal C-terminal end, which possibly leads to the escape of NMD. Notably, the C-terminal region of CUX1 has a regulatory function over the transcriptional activity of the p200 protein, and Caspase-L protease can cut it during the cell cycle. This cleavage increases the transcriptional activity of the CUX1 p200 protein [29]. Hence, the variants of individuals 1, 26, 28, and 29 could lead to proteins with altered transcriptional activity compared to WT CUX1. Individual 34 had an inversion with breakpoints within intron 20 and the 3’UTR of exon 24. Although it is unclear whether this variant leads to NMD, this allele likely causes impaired CUX1 activity.
Regarding the missense variants, three of these affect highly conserved amino acid residues. However, in silico predicting programs render only slightly increased scores. Furthermore, the de novo missense variant c.4064C > T (p.(Thr1355Ile)) affects a weakly conserved residue predicted to be benign by several in silico prediction programs (Table S2).
Analysis of Cux1 heterozygous mice
To investigate the underlying mechanisms of the Cux1 haploinsufficiency, we characterized a previously described mouse line that carries a truncating deletion in the homeodomain of Cux1. Although there were no previously reported alterations in the development of these heterozygous animals [13], we observed a slight reduction of early postnatal growth (Fig. S5A). Histological analysis of brain structures revealed no differences between Cux1+/− and WT animals (Fig. S5B).
We next quantified Cux1 transcripts in WT and Cux1+/− mouse cortices using RT-qPCR. This analysis evaluated the expression only of the WT isoforms and not the mutant transcripts (see Fig. 2A, B). As the clinical course of individuals suggests different impacts of heterozygosity at distinct developmental stages, we analyzed transcripts in the cortex of both young postnatal (P10) and mature (P30) animals. Cux1 transcripts were reduced to half the levels of WT, both in P10 and P30 Cux1+/−cortices (Fig. 2C). As expected, as Cux1 null alleles affect exons not present in CASP, we found no changes in the expression of CASP transcripts (Fig. 2D). This indicates that the expression of the WT allele is not upregulated to compensate for the null allele in Cux1+/− mice.
A Exon structure of Cux1 genomic sequence and detail showing the variant deleting exons 23 and 24 in Cux1+/−. Vertical lines represent individual exons. Arrows highlight primer sequences used for the quantifications of WT transcripts by RT-qPCR. B Predicted transcripts coding for CUX1 and CASP. Boxes and dashed boxes highlight the regions containing the RT-qPCR amplicons used to quantify protein-coding transcripts. The Cux1 amplicon measures all annotated CUX1 protein-coding transcripts (Cux1-201, 204, 209, 212, 206) except Cux1-208. RT-qPCR amplicon for CASP measures all annotated CASP protein-coding transcripts (Cux1-211, 207, 205). C Relative expression of Cux1 protein-coding transcript isoforms (Cux1-201, 204, 209, 212, 206) as shown in (A) and (B) at P10 and P30, quantified by RT-q-PCR. Data are shown normalized to P10 WT levels. Data show mean ± SEM (n ≥ 3 animals per condition. Two-way ANOVA: P-value WT vs. Cux+/- #### ≤0.0001. Post hoc with Tukey´s test: P-value P10 WT vs. Cux1+/− ** ≤0.005, P-value P30 WT vs. Cux+/−** ≤0.005). D Relative gene expression of protein-coding CASP transcripts at P10 and P30. Data are shown normalized to P10 WT levels. Data show mean ± SEM (n ≥ 3 animals per condition. Two-way ANOVA: P-value WT vs. Cux+/- # ≤0.05. Post hoc with Tukey´s test: P-value P10 WT vs. Cux+/− = 0.4641 (n.s.), P-value P30 WT vs. Cux+/− = 0.1846 (n.s.)).
To investigate CUX1 expression in the cortex of heterozygous mice, we immunostained brain coronal sections of P10 and P30 animals and quantified CUX1 levels in several cortical areas and layers, using an antibody against the C-terminal region of CUX1 but not CASP (Fig. 3). We observed a significant reduction of CUX1 immunoreactivity in P10 Cux1+/− mice compared to WT animals, especially in L4 neurons (Fig. 3C, D). In contrast, there was no significant difference of CUX1 immunoreactivity in P30 brains (Fig. 3E, F).
A Comparative scheme of distinct functional areas in human (top) and mouse (bottom) brains. Top, dorsal (left), and lateral (right) views of motor (MO, green), somatosensory (SS, blue), auditory (AUD, magenta), and temporal association (TeA) cortical areas in the human brain. Bottom, dorsal (left), and medio-lateral views at several anteroposterior coordinates (right) of the functional areas in the mouse brain. B Intensity maps of Cux1 expression early in development (P10) in WT and Cux1+/− mouse brains. Images show coronal sections from more anterior (left) to more posterior (right) coordinates. Dashed boxes in the middle images highlight areas of interest (SSbf, somatosensory barrel field). Scale bar = 500 µm. C, E Magnified images of cortical upper-layer neurons (L2-4) from dashed areas of Fig. S1B at P10 (C) and P30 (E). Scale bar = 200 µm. D, F Quantification of Cux1 expression in upper-layer neurons of SS, SSbf, and TeA areas at P10 (D) and P30 (F). Data show mean ± SEM (n ≥ 3 animals per condition, n = 2 sections per brain. P10 Two-way ANOVA: P-value WT vs. Cux1+/− #### ≤ 0.0001. Post hoc with Sidak´s test: P-value SSL4 WT vs. Cux1+/− ** ≤ 0.01, P-value SSbfL4 WT vs. Cux+/− *** ≤ 0.001, P-value TeAL4 WT vs. Cux+/− ** ≤ 0.01. P30 Two-way ANOVA: P-value WT vs. Cux1+/−## ≤ 0.01.). G Western blot showing cortical expression of the full-length 200 kDa CUX1 in WT, Cux1+/−, and Cux1−/− (E18 only) mice at E18, P10, P30, and P135. The amount of protein was quantified and normalized to α tubulin expression (50 kDa). CASP (75 kDa), an alternatively spliced product of the Cux1 gene, is also recognized by this antibody. The truncated mutant CUX1 is indicated by an arrowhead. H Relative cortical expression of the 200 kDa CUX1 at P10, P30, and P135. Data show mean ± SEM. (n = 3–4 cortical samples per condition. P10 unpaired t test: P-value WT vs. Cux1+/− * ≤ 0.05. P30 unpaired t test: P-value WT vs. Cux1+/− ** ≤ 0.01. P135 unpaired t test: P-value WT vs. Cux+/− *** ≤ 0.001).
As different CUX1 isoforms show different transcriptional activity [1, 4], we analyzed protein expression using western blot. Of note, the antibody used for immunofluorescence is unfortunately unsuitable for western blot. The available antibody for immunoblotting recognizes the common N-terminal region included in p200 CUX1 and CASP but not the shorter CUX1 isoforms. This analysis demonstrated a significant reduction in the expression of p200 CUX1 in Cux1+/− mice compared to WT at all tested ages (Fig. 3G, H). The blots also confirmed that CASP expression is unaffected in Cux1+/− mice (Fig. 3G). As control of antibody specificity, we confirmed the absence of the p200 CUX1 band in lysates from E18 Cux1−/− embryos. In both Cux1−/− and Cux1+/− cortices, we also detected low levels of the mutant truncated CUX1 reported in previous studies [13] (Fig. 3G).
Thus, western blots showed equal reductions of p200 CUX1 expression at all ages. At the same time, immunofluorescence indicated a more significant deficiency of total CUX1 during development than in adulthood. These observations suggest that the lower immunofluorescence reductions in adults are likely due to immunoreactivity from short CUX1 isoforms. As P10 and P30 Cux1+/− mice show decreases in all transcripts, it is conceivable that post-transcriptional mechanisms balance the amount of shorter CUX1 isoforms in older animals by proteolysis of p200 CUX1.
Finally, as eight individuals in the cohort had seizures, we analyzed the susceptibility of Cux1+/− mice to seizures upon administration of kainic acid (Fig. 4A). Only Cux1+/− and not WT animals developed severe attacks (Fig. 4B). There was, however, no difference in latency to the intermediate epileptic stage R3 (Fig. 4C). Thus, correlating with clinical findings, Cux1+/− mice demonstrated increased epileptic susceptibility compared to WT.
A Identification of stages of seizure in a kainate-induced model of epilepsy. Photographs show representative behaviors from the less severe to the most, progressing from non-convulsive to convulsive seizure stages (R1-R6). B The maximum stage of seizure was reached in WT and Cux1+/− mice during the first 2 h of kainate induction. Data show mean ± SEM (n = 10 per condition. Mann–Whitney test: P-value WT vs. Cux1+/− ** ≤ 0.01). C Latency to the onset of convulsive stages (R3) in WT and Cux1+/− mice after kainate induction. Data show mean ± SEM (n ≥ 8 per condition. Mann–Whitney test: P-value WT vs. Cux+/− = 0.3478 (n.s.)).