Extending the phenotypes associated with TRIO gene variants in a cohort of 25 patients and review of the literature

The TRIO gene encodes a rho guanine exchange factor, the function of which is to exchange GDP to GTP, and hence to activate Rho GTPases, and has been described to impact neurodevelopment. Specific genotype‐to‐phenotype correlations have been established previously describing striking differentiating features seen in variants located in specific domains of the TRIO gene that are associated with opposite effects on RAC1 activity. Currently, 32 cases with a TRIO gene alteration have been published in the medical literature. Here, we report an additional 25, previously unreported individuals who possess heterozygous TRIO variants and we review the literature. In addition, functional studies were performed on the c.4394A > G (N1465S) and c.6244‐2A > G TRIO variants to provide evidence for their pathogenicity. Variants reported by the current study include missense variants, truncating nonsense variants, and an intragenic deletion. Clinical features were previously described and included developmental delay, learning difficulties, microcephaly, macrocephaly, seizures, behavioral issues (aggression, stereotypies), skeletal problems including short, tapering fingers and scoliosis, dental problems (overcrowding/delayed eruption), and variable facial features. Here, we report clinical features that have not been described previously, including specific structural brain malformations such as abnormalities of the corpus callosum and ventriculomegaly, additional psychological and dental issues along with a more recognizable facial gestalt linked to the specific domains of the TRIO gene and the effect of the variant upon the function of the encoded protein. This current study further strengthens the genotype‐to‐phenotype correlation that was previously established and extends the range of phenotypes to include structural brain abnormalities, additional skeletal, dental, and psychiatric issues.

TRIO gene missense variants have been shown to cluster in two main mutational hotspots, namely the spectrin and GEFD1 domains, which are linked to two distinct phenotypes. Pathogenic and likely pathogenic variants within the spectrin domain have been associated with more severe developmental issues and macrocephaly, via TRIOinduced RAC1 hyperactivity and a gain-of-function mechanism, whereas variants in the GEFD1 domain were shown to lead to a milder phenotype with less pronounced neurodevelopmental delay and microcephaly, with demonstrable reduced activity of the TRIO protein and a loss-of-function mechanism (Barbosa et al., 2020).
Here, we describe detailed phenotype information of an additional 25 patients and review previously published cases. The current study extends the spectrum of phenotypes with additional skeletal features, dental anomalies, and structural brain malformations. In addition, psychiatric issues including bipolar disorders and anxiety are described.
This is the largest cohort of patients with a TRIO variant to date, with a potential to extend and refine the phenotypic associations with mutations in this gene.

| PATIENTS AND METHODS
Patients and families wishing to participate in the current project contacted our team via a patient support group called Team TRIO (www. teamtrio.org) between July 2021 and August 2022. We also included

| FUNCTIONAL STUDY ON TRIO VARIANT N1465S
HEK293T and N1E-115 neuroblastoma cells were cultured and transfected as described in the study by Barbosa et al. (2020). Immunoblot analysis for quantification of Phospho-PAK levels was also performed as described in the study Barbosa et al. (2020) (Barbosa et al., 2020). As shown in Figure 1a,b, the N1465S variant impaired the ability of TRIO to activate RAC1, as phosphoPAK levels were significantly reduced, similarly to the GEF dead control. To confirm this effect, we used the N1E-115 neuroblastoma cell line, in which TRIO expression leads to enhanced neurite outgrowth, in a RAC1-dependent manner. As shown in Figure 1c, the N1465S variant was impaired in inducing neurite outgrowth. These two data allowed us to reclassify the p.Asn1465Ser VUS into the likely pathogenic category.
In addition, the pathogenicity of the variant c.6244-2A > G identified in Patient 23 was confirmed by splicing analysis. This has shown aberrant alternative acceptor site activation causing a 26 base pair loss and subsequent frame shift confirming pathogenicity. This result was confirmed by Sanger sequencing (see Figure S1).

| Behavioral/psychiatric phenotype
Information regarding behavioral issues was provided in four out of the seven participants and two of the four (50%) had stereotypies, three of the four (75%) exhibited aggressive behavior, three of the four (75%) were described as having poor attention and two of the four (50%) had OCD traits. One of the seven cases had autism (Patient 11) and one of seven (Patient 15) had hyperactivity (Table 2).

| Gastrointestinal, skeletal, dental, and other features
Two of the four (50%) individuals were reported to have infantile feeding difficulties with one of the two cases requiring tube feeding.
In addition, three of the four (75%) cases had constipation and three of the four (75%) cases had growth retardation.
Skeletal features were observed in this group of patients with two of the four (50%) having scoliosis, three of the four (75%) cases having short tapering fingers, and two of the four (50%) 2-3 toe syndactyly. Pectus excavatum, pes planus, and hypermobility were observed in single cases. Dental overcrowding was seen in one of the four cases (Patient 10) and the same individual (Patient 10) had delayed dental eruption (Table 2).

| Brain malformations
Structural brain abnormalities were described in two cases and included hypoplasia or agenesis of corpus callosum in one participant (Patient 10). Patient 10 also had dysmorphic lateral ventricles and dysmorphic basal ganglia. In addition, one patient (Patient 16) had gliotic scarring in the right middle cerebral artery (MCA) and duplication of the inferior branch of the right MCA and a further individual (Patient 13) had syrinx of the spine (Table 2).

| Facial characteristics
Photographs of five of the seven cases were made available, which revealed a recognizable facial gestalt including microcephaly with hypertelorism, upslanting palpebral fissures, synophrys; long tubular nose with a bulbous nasal tip and thin upper lip (Figure 3).

| Gastrointestinal, skeletal, dental, and other features
Infantile feeding problems were present in seven of the nine (78%) cases including the patient with the intragenic deletion with only the latter and another patient requiring tube feeding. Constipation was recorded in two of the nine (22%) cases and five out of the nine (55%) cases were described as having growth retardation. Skeletal features did not appear to be common in this group of patients, with short, tapering fingers described in two cases with TV; one of these individuals (Patient 20) also had a short fifth toe and short nails.

| Brain abnormalities
Structural brain malformation was seen in four cases (Patients 18,19,23,and 24) and these included ventriculomegaly, lissencephaly, global cerebral atrophy, secondary agenesis of corpus callosum, and progressive leucoencephalopathy on the MRI brain scan (Table 3).

| Facial characteristics
Photographs were provided for six of the nine cases and clinical features appeared to resemble those seen in individuals with missense variants in the GEFD1/2 domain. These included microcephaly, upslanting palpebral fissures, long, tubular nose with a bulbous nasal tip and thin upper lip (Figure 4).

| DISCUSSION
The TRIO gene encodes a protein that exchanges GDP to GTP on Rho GTPases and has a role in the remodeling of the cytoskeleton, hence impacting on cell migration and growth (Schmidt & Debant, 2014). Comparing the 25 cases presented here with those reported previously (see Table 4), there appears to be a recognizable split between clinical features of individuals with a missense variant in the spectrin domain and those with a missense variant in the GEFD1 or GEFD2 domains, truncating variants or deletions.

| Missense variants in the spectrin domain
All nine patients presented here were delayed in the acquisition of their developmental milestones ranging from moderate to severe, which is in keeping with findings from previous studies. Short stature was described in three cases here and growth retardation in five of the nine cases, in addition to one case that was published by Pengelly et al. (Pengelly et al., 2016). All cases, where measurements of the head circumference were available, had absolute-or in a few cases relative-macrocephaly, which is identical to TRIO cases in other case series with this finding. Neurobehavioral phenotypes were previously described in the study by Pengelly et al. (2016) paper and included stereotypies, aggression, and poor attention span. Barbosa et al. (Barbosa et al., 2020) also described an overall incidence of 36% for aggression, 27% for stereotypies, 45% OCD traits, 31% autistic traits, and 70% had poor attention in the TRIO cohort. In our spectrin cohort, the rate of stereotypies was 33.3% (3/9), aggression was 33.3% (3/9), poor attention was 55.5% (5/9), and OCD traits were 43% (3/7). In addition, hyperactivity, autism, and ADHD were described as in previous papers. Seizures occurred in four of the nine cases (44.4%), which is similar to those seen in the Barbosa paper (33.3%). Infantile feeding difficulties were delineated in the spectrin case of the Pengelly paper and found in 55.5% (5/9) in the cases here and constipation was seen Neurobehavioral phenotypes were described in the current cohort (including patients with GEFD1/2 variants, TVs, and the intragenic deletion) with stereotypies (4/13, 33.3%), aggression (6/13, 46%), poor attention (10/13, 77%), OCD traits (5/13, 38%), and seizures (4/13, 31%) being described. These features were also delineated in the Barbosa study, although only an overall figure was presented for the TRIO cohort there. Of note, neurobehavioral phenotypes appeared to be slightly less common in the TV group (including the intragenic deletion) with the exception of poor attention that was seen in seven of the nine (78%) cases (Table 4). Infantile feeding difficulties (9/13, 61%), tube feeding (3/13, 23%), and constipation ( in the GEFD1/GEFD2 and TVs are evident and could be explained by the effect of the GEFD1 variants on RAC1 leading to reduced activity of the TRIO protein and a loss-of-function mechanism as shown by Barbosa et al. (2020).
The current study further supports the finding that missense variants in the spectrin domain cause more significant developmental delay with prominent neurological and behavior features including seizures, stereotypies, autism, OCD, and hyperactivity, while frameshift and GEFD1/2 variants cause a milder phenotype with mild delay but also with significant neurological and behavior involvement. Difference in the phenotype was not observed in gastrointestinal, dental, and skeletal phenotypes.
In view of these findings, we recommend neurodevelopmental, dental, and spine surveillance for individuals with TRIO gene variants, with brain MRI performed at their initial assessment. In addition, gastrointestinal, psychiatric and behavioral assessments are recommended.
Further studies are required to determine the possibility of longterm complications related to TRIO and to uncover the significance of the structural brain malformations and the psychiatric issues.
In summary, this case series brings the number of reported TRIO cases to 57 patients with either a variant in spectrin and a likely gain-   Growth ret.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request. These include the data collection forms and details of the functional studies.