A de novo FOXP1 variant in a patient with autism, intellectual disability and severe speech and language impairment

Abstract

FOXP1 (forkhead box protein P1) is a transcription factor involved in the development of several tissues, including the brain. An emerging phenotype of patients with protein-disrupting FOXP1 variants includes global developmental delay, intellectual disability and mild to severe speech/language deficits. We report on a female child with a history of severe hypotonia, autism spectrum disorder and mild intellectual disability with severe speech/language impairment. Clinical exome sequencing identified a heterozygous de novo FOXP1 variant c.1267_1268delGT (p.V423Hfs*37). Functional analyses using cellular models show that the variant disrupts multiple aspects of FOXP1 activity, including subcellular localization and transcriptional repression properties. Our findings highlight the importance of performing functional characterization to help uncover the biological significance of variants identified by genomics approaches, thereby providing insight into pathways underlying complex neurodevelopmental disorders. Moreover, our data support the hypothesis that de novo variants represent significant causal factors in severe sporadic disorders and extend the phenotype seen in individuals with FOXP1 haploinsufficiency.

Figure 1

Identification of a de novo FOXP1 variant in a patient with hypotonia, autism, mild ID and speech/language deficits. (a) Photograph of the patient at 14 years of age showing macrocephaly, mild ptosis, wide-spaced teeth and a hypotonic face. (b) Family pedigree showing Sanger traces of genomic DNA from the patient and one unaffected parent. The affected proband (shaded symbol) carries a heterozygous de novo two-base deletion (-GT relative to mRNA; c.1267_1268delGT) that introduces a frameshift and results in a premature stop codon. Site of deletion is indicated by black arrow. (c) Schematic representation of the FOXP1 protein indicating the change found in the patient. The FOXP1 protein has a glutamine-rich (Q-rich) region and zinc-finger (ZF), leucine zipper (LZ) and FOX DNA-binding domains, as well as two nuclear localization signals (NLS1 and NLS2). The change in FOXP1 is predicted to yield a C-terminally truncated protein (p.V423Hfs*37) that lacks the FOX DNA-binding domain and NLS. Other truncated FOXP1 variants previously reported in ASD (p.A339Sfs*4) and ID (p.R525*) are also shown. (d) Sanger traces of FOXP1 cDNA from immortalized lymphoblasts derived from the affected proband and controls (father, mother and unaffected sibling). Two sets of cultures were grown in parallel, one of which was treated with 100 μg/ml emetine for 7 h to inhibit NMD. In untreated cells, there is no evidence of the variant transcript, suggesting that the majority of these transcripts are degraded by NMD. Sanger traces from left to right: father, mother, proband, unaffected sibling. Arrows indicate site of deletion. (e) Quantitative RT-PCR amplification of variant FOXP1 transcripts in cDNA derived from the proband and controls (father, mother and unaffected sibling) (left panel). Melting curve analysis was performed to assess the specificity of the amplification (right panel). Primers were designed to specifically detect the variant allele and amplification of c.1267_1268delGT was only seen in proband-derived cDNA (black lines). Results represent two technical replicates per condition. POLR2F was used as a control. RFU, raw fluorescence unit.

Figure 2

Functional characterization of the de novo FOXP1 variant. (a) Immunoblot of FOXP1 proteins fused to YFP in transfected HEK293 cells demonstrates similar levels of expression between WT FOXP1 and the mutant protein. Control denotes cells transfected with empty pYFP vector. Equal protein loading was confirmed by stripping and reprobing with an anti-β-actin antibody. (b) Fluorescence imaging of HEK293 cells transfected with WT or mutant FOXP1 fused to mCherry (red). The p.V423Hfs*37 variant displays aberrant localization to the cytoplasm. Nuclei were stained with Hoescht 33342 (blue). Scale bar=10 μm. Concordant results were seen in SHSY5Y cells, as shown in Supplementary Figure 3. (c) Luciferase reporter assays for transcriptional regulatory activity of FOXP1 variants in HEK293 cells. The FOXP1 p.V423Hfs*37 variant failed to repress expression of the luciferase reporter, indicating loss of transcriptional repressor activity. Values are expressed relative to the control (***P<0.001; NS, not significant). The mean ±SEM of three independent experiments performed in triplicate is shown. (d) BRET assays for interaction between the p.V423Hfs*37 variant and WT FOXP proteins in live HEK293 cells. Bars represent the corrected mean BRET ratios±SEM of one experiment performed in triplicate. (e) Fluorescence images of HEK293 cells transfected with WT and mutant FOXPs. In the absence of the p.V423Hfs*37 variant (fused to mCherry; red), FOXP1 and FOXP2 are nuclear (fused to YFP; green) (left panel). When coexpressed with the p.V423Hfs*37 variant, FOXP1 and FOXP2 mislocalize to the cytoplasm (right panel). Nuclei were stained with Hoescht 33342 (blue). Scale bar=10 μm. Concordant results were seen in SHSY5Y cells, as shown in Supplementary Figure 3.