Background
Haploinsufficiency of the forkhead-box protein P1 (
FOXP1) gene has recently been shown to cause a neurodevelopmental disorder with a phenotype characterized by global developmental delay (DD), intellectual disability (ID), speech deficits, mild dysmorphic features, and autism spectrum disorder (ASD) traits [
1‐
5]. Here, we refer to this disorder as FOXP1 syndrome.
Since the first report of a deletion spanning
FOXP1 and three other genes in a child with DD, speech delay, hypertonia, dysmorphic features, contractures and blepharophimosis [
6], nearly 20 cases have been reported. The identification of deletions limited to
FOXP1 [
1‐
3,
7,
8] and of several individuals with loss-of-function and missense variants in
FOXP1 [
1,
3‐
5,
9,
10] has delineated
FOXP1 haploinsufficiency as sufficient to produce core features including delayed motor and language milestones, global speech impairment, ID, dysmorphic features, and ASD. Additional
FOXP1 mutations have also emerged from large-scale targeted sequencing or exome and genome sequencing analyses of cohorts with ID [
11‐
13] or ASD [
14,
15]; however, these studies have provided minimal phenotypic information.
FOXP1 can be among the genes deleted in cases with 3p14 deletion syndrome, a contiguous gene syndrome that also presents with hearing loss, congenital heart defects, and urogenital abnormalities [
16‐
18]. Interestingly, common variation at the
FOXP1 locus has shown association in a cross-disorder meta-analysis of ASD and genome-wide association studies in schizophrenia [
19]. Eight additional pathogenic mutations have emerged after the discovery of a de novo mutation in a whole exome sequencing (WES) study on congenital anomalies of the kidney and urinary tract [
20]. Notably, all individuals have neurodevelopmental phenotypes compatible with FOXP1 syndrome and the majority (6/8) display upper or lower urinary tract defects [
20].
FOXP1 is a transcription factor of the
FOX gene family, named for the forkhead-box DNA-binding domain present in the gene family [
21]. The
FOXP subfamily is comprised of four genes:
FOXP1,
FOXP2,
FOXP3, and
FOXP4. The closest homolog to
FOXP1, and the best-known member of the
FOXP family, is
FOXP2. In the brains of zebra finch songbirds and humans, Foxp1 and Foxp2 are co-expressed in the GABAergic medium spiny neurons of the striatum [
22,
23], a brain region critical for human language, mouse ultrasonic vocalization (USV), and zebra finch vocal imitation. Both genes are important for language production and comprehension. In addition to the speech impairments observed in individuals with FOXP1 syndrome, maternal uniparental disomy of chromosome 7 (reducing
FOXP2 expression) [
24],
FOXP2 deletions [
25], and
FOXP2 mutations [
26,
27], all result in childhood apraxia of speech and other speech and language defects. In addition, both
FOXP1 and
FOXP2 are critical during cortical neurogenesis and specification [
28‐
30]. Constitutive
FOXP1 knockout is embryonically lethal due to a cardiac defect, but brain-specific conditional
FOXP1 null mice display striatal morphological defects, reduced USV (also reported in
FOXP2 mutant mice [
31]) and social and cognitive deficits [
23,
32].
Shared and pervasive clinical features in individuals with
FOXP1 deletions and mutations include mild-to-moderate ID, language impairment, and motor delays [
1‐
5,
10,
13]. All reported individuals with FOXP1 syndrome displayed speech and language impairment. While delayed, the developmental trajectory of cognitive, language, and motor skills remains unknown. The majority of individuals described in the literature developed a minimum of phrase speech, all individuals were reported to have difficulty with articulation, and language was often limited to phrases or simple sentences [
1,
2,
4,
5]. Several investigators reported that expressive language is more affected than receptive language [
1‐
3]; however, these findings were not based on norm-referenced standardized testing.
Medical features of individuals with
FOXP1 mutations reported in the literature vary widely and include brain and cardiac malformations, hypotonia, strabismus, and obesity [
2,
4,
5,
7,
16,
33,
34]. Dysmorphic features associated with FOXP1 syndrome appear to be mild and inconsistent. Among the most commonly reported dysmorphic features are a prominent forehead, downslanting or short palpebral fissures, and a short nose with a broad tip [
3]. Other features may include widely spaced eyes, frontal hair upsweep, ptosis, and hypertelorism [
7]. Behavioral anomalies, including ASD or autistic traits, aggression, anxiety, and obsessive-compulsive symptoms, were present in a majority of reported cases [
1,
4,
5,
8,
10,
13].
To date, no study has prospectively evaluated more than three individuals with FOXP1 syndrome using a battery of standardized measures. The goal of the present study was to comprehensively characterize FOXP1 syndrome by utilizing a multidisciplinary team of biologists and clinicians and objective assessments to prospectively evaluate a cohort of children and adolescents with mutations in the
FOXP1 gene. We also characterize one individual with a duplication of 8.4 Mb spanning
FOXP1 and 47 additional genes, which has not previously been described and remains of unknown clinical significance (Additional file
1). Evaluating genetic results in conjunction with a robust battery of clinical assessments will better elucidate the genotype-phenotype relationship in this recently described syndrome, while functional dissection of mutations will provide insights into the pathobiological mechanisms underlying the FOXP1 syndrome.
Discussion
In this study, we report on the genetic and clinical spectrum of FOXP1 syndrome in a cohort of nine individuals with mutations in FOXP1 and one individual with a large duplication spanning FOXP1, evaluated as of unknown significance.
Few individuals have been described in the literature, but screening the emerging
FOXP1 mutational landscape reveals 34 private mutations and another 7 that recur in unrelated individuals and appear as mutation hotspots in the gene (Fig.
1). The identification of recurrent mutations has important implications for clinical genetics practice and offers the opportunity to evaluate clinical variability in FOXP1 syndrome. For example, individual S1 has average cognitive functioning, although she carries a mutation as disruptive as other loss-of-function mutations in our cohort (Fig.
2).
Another key observation emerging from our analyses is that over 80% of the pathogenic missense mutations, including all four missense mutations in our cohort, lie in the DNA-binding domain and perturb amino acids that are necessary for the binding to the DNA or to the domain swapping that mediates FOXP1 dimerization (Fig.
3). This finding emphasizes the importance of carefully evaluating
FOXP1 missense variants and taking into account structural information when evaluating pathogenicity.
Our clinical observations delineate a clinical spectrum of FOXP1 syndrome that includes a set of core phenotypic features, including delays in early motor and language milestones, language impairment, ASD symptoms (although subthreshold for a DSM-5 diagnosis in the majority of individuals), and visual-motor integration deficits (Table
2). Psychiatric features were also prominent including anxiety, obsessive-compulsive traits, attention deficits, and externalizing symptoms. Cognitive ability ranged from profound ID to average, with the majority of individuals performing in the range of mild ID (standard scores between 50 and 70). Interestingly, there was not a significant difference between verbal and nonverbal abilities. Adaptive functioning was similarly developed, suggesting evenly developed skills.
With regard to ASD symptoms, the majority of individuals fell above ASD cutoffs on standardized diagnostic assessments (ADOS-2, ADI-R); however, only two individuals met DSM-5 criteria for ASD (Additional file
3: Table S2). It is notable that a greater number of symptoms were observed in the Restricted and Repetitive Behavior domain as compared to the Social Communication domain. While results highlight the role of clinical judgment and suggest that a high level of expertise is required to fully assess ASD, individuals with FOXP1 syndrome will nevertheless likely benefit from similar treatments to those with ASD. In addition to interventions targeting repetitive behaviors, sensory symptoms, and compulsive-like behaviors, social skills training is likely warranted given the extent of social difficulties endorsed by caregivers across measures.
In contrast to findings from previous studies [
1‐
3], our results using norm-referenced language assessments indicate that expressive language is better developed than receptive language. Parents reported fewer skills in both expressive and receptive subdomains on the Vineland-II as compared to results from clinician-administered assessments (Expressive Vocabulary Test, 2nd Edition and Peabody Picture Vocabulary Test, 4
th Edition). This discrepancy likely reflects differences between language ability and the application of language during daily functioning. Visual-motor integration and motor coordination deficits were also present in all individuals. Language and motor weaknesses appear to emerge early, as evidenced by delays in the achievement of developmental milestones.
The wide age range of participants did appear to impact performance on standardized assessments, particularly cognitive testing. Within this cohort, the two youngest participants achieved the highest IQ scores and the oldest participant achieved the lowest IQ score. As individuals with developmental delays age, scores on standardized testing often declines due to an individual’s failure to gain new skills at the expected pace. Larger samples are needed to assess the progression of the syndrome and to better understand the variability in clinical presentation across patients.
The individual with the
FOXP1 duplication presented with a similar phenotype, which included delays in reaching developmental milestones, borderline cognitive and adaptive functioning, visual-motor integration deficits, sub-threshold ASD symptoms, and clinically significant levels of anxiety (Additional file
1).
On the neurological exam, fine and gross motor coordination deficits were present in all individuals, which is consistent with motor delays and continued deficits in the motor domain. Structural abnormalities on brain MRI were detected in the majority of individuals but did not follow a specific pattern. Enlarged ventricles were the most common imaging finding and this has been reported previously [
3,
20]. There was no report of seizure disorder in any individual.
On the medical exam, we observed congenital heart defects in two out of six individuals examined. An association between congenital heart defects and
FOXP1 haploinsufficiency has been suggested in an earlier report [
63] and sporadically detected in affected individuals [
20], but not replicated in a large-scale WES study on congenital heart defects [
64]. Nevertheless,
FOXP1 plays a key role in cardiac morphogenesis in mice [
65] and cardiac problems should be assessed in individuals with
FOXP1 mutations. Another relevant medical finding is related to congenital anomalies of the kidney and urinary tract. A previous study reported eight individuals with de novo mutations in FOXP1, six of which had congenital anomalies of the kidney and urinary tract [
20]. One individual in our cohort had congenital renal defects. While genitourinary abnormalities were not reported in the other patients in our cohort, it remains important to consider kidney/urinary tract congenital anomalies when assessing individuals with FOXP1 syndrome. In addition, constipation was reported by several parents in our cohort and is often present in children with neurodevelopmental disorders, especially in conditions associated with hypotonia. On review of the medications used in our cohort, it does not appear that the extent of constipation can be accounted for by psychotropic medication use. The majority of individuals affected by constipation were either not receiving medications, or not receiving medications where constipation is a common side effect.
On the dysmorphology exam, although non-specific, over half the cohort presented with several dysmorphic features including a broad nasal bridge, prominent forehead, bulbous nose, high arched palate, clinodactyly, strabismus, and hypertelorism (Fig.
4).
Our genetic findings can also inform the design of patient-specific cellular models and animal models with stronger construct validity, which are at this point critical to understand the underpinnings of FOXP1 syndrome. Thus far, cultured rodent neurons have been employed to identify the defects in neuronal morphology and physiology resulting from
FOXP1 silencing [
66] or knockout [
32]. Also, the functional consequences of
FOXP1 mutations have been investigated only in non-neuronal cells.
FOXP1 mRNA harboring loss-of-function mutations are likely to undergo non-sense mediated decay, as shown for p.Ala339Serfs4* [
10], but at least a fraction of them escape non-sense mediated decay. Exogenously expressed FOXP1 mutants harboring p.Val423Hisfs*37 [
4], p.Ala339Serfs4* [
5,
10], p.Tyr439*, or p.Arg525* [
5] have been shown to disrupt nuclear localization. Missense mutations result in aberrant aggregates in the nucleus and cytoplasm (p.Arg514Cys and p.Arg465Gly) or only in the cytoplasm (p.Trp534Arg) [
5]. These eight mutations abolish the transcriptional repression activity of FOXP1, as shown by enhanced expression of a luciferase reporter [
1,
4,
5]. While mutations p.Ala339Serfs4*, p.Arg525*, and p.Trp534Arg suppress the interaction with wildtype FOXP1 and FOXP2 [
4,
5], FOXP1 mutants carrying p.Tyr439*, p.Arg514Cys, or p.Arg465Gly retain the ability to bind FOXP1 and FOXP1 and might exert dominant-negative effects [
5]. Investigating neuronal models carrying recurrent pathogenic mutations or patient-derived human neuronal models is key to comprehensively expose the pathophysiological mechanisms underlying FOXP1 syndrome.
Similarly, mouse models studied thus far have brain-specific knockout of
FOXP1 [
23,
32]. While these models have been instrumental in elucidating the fundamental role of
FOXP1 in the striatum and their relevance to some of the phenotypes observed in individuals, they recapitulate only in part the genetic architecture of FOXP1 syndrome. First, they accurately model only cases with
FOXP1 deletions rather than mutations. A strategy to mimic patient-specific mutations has been applied for
FOXP2 by generating a knockin mouse with the equivalent of the p.Arg553His human mutation (p.Arg514His in
FOXP1) [
67]. Second, these studies have used homozygous animals, while the syndrome results from a heterozygous defect, rather than complete loss of
FOXP1. The clinical evidence reported here might inform targeted phenotypic characterization of
Foxp1 rodent models to fully capture the clinical features of the syndrome, including the neurodevelopmental phenotype and the other medical co-morbidities. Based on the clinical phenotypes in individuals with FOXP1 syndrome, a comprehensive characterization of heterozygous
FOXP1 rodent models should include an examination of motor abnormalities, hyperactivity and executive functioning, repetitive and compulsive behaviors, social and communication deficits, cognitive changes, anxiety, and circadian/sleep abnormalities. Deficits in visual-motor integration would be particularly interesting to study. In addition, careful examination of cardiac, renal and visual organs, as well as brain ventricle size would be warranted. Finally, immune and endocrine function should be investigated.