De novo and inherited TCF20 pathogenic variants are associated with intellectual disability, dysmorphic features, hypotonia, and neurological impairments with similarities to Smith–Magenis syndrome

The human chromosome 22q13 region is involved with various genetic and genomic disorders, including Phelan–McDermid syndrome (MIM 606232), in which terminal deletion of 22q13.3 encompassing the critical gene SHANK3 is frequently observed [1]. Occasionally, deletions proximal to the classical Phelan–McDermid syndrome region have been reported, affecting chromosome 22q13.2 without directly disrupting SHANK3 [2–4]. It remains unknown whether the abnormal neurodevelopmental phenotypes observed in patients with 22q13.2 deletions are caused by dysregulation of SHANK3 or haploinsufficiency of previously undefined “diseases genes” within the deletion. Recently, a bioinformatics analysis of genes within 22q13.2 highlighted that TCF20 and SULT4A1 are the only two genes within this region that are predicted to be highly intolerant to loss-of-function (LoF) variants and are involved in human neurodevelopmental processes [5]. In particular, TCF20 was predicted to be of higher intolerance to LoF variants as reflected by its higher pLI (probability of LoF intolerance) score (pLI = 1), making it the most promising candidate disease gene underlying neurodevelopmental traits associated with 22q13.2 deletion disorders.

TCF20 (encoding a protein previously known as SPRE-binding protein, SPBP) is composed of six exons, which encode two open reading frames of 5880 or 5814 nucleotides generated by alternative splicing. The shorter isoform (referred to as isoform 2, Genbank: NM_181492.2) lacks exon 5 in the 3′ coding region. Isoform 1 (Genbank: NM_005650.3) is exclusively expressed in the brain, heart, and testis and predominates in the liver and kidney. Isoform 2 is mostly expressed in the lung ([6, 7]; Fig. 1). TCF20 was originally found to be involved in transcriptional activation of the MMP3 (matrix metalloproteinase 3, MIM 185250) promoter through a specific DNA sequence [8]. More recently, it has been shown to act as a transcriptional regulator augmenting or repressing the expression of a multitude of transcription factors including SP1 (specificity protein 1 MIM 189906), PAX6 (paired box protein 6, MIM 607108), ETS1 (E twenty-six 1, MIM 164720), SNURF (SNRPN upstream reading frame)/RNF4 (MIM 602850), and AR (androgen receptor, MIM 313700) among others [9–11]. TCF20 is widely expressed and shows increased expression in the developing mouse brain particularly in the hippocampus and cerebellum [12, 13]. Babbs et al. studied a cohort of patients with autism spectrum disorders (ASDs) and proposed TCF20 as a candidate gene for ASD based on four patients with de novo heterozygous potentially deleterious changes, including two siblings with a translocation disrupting the coding region of TCF20, one frameshift and one missense change in another two patients [6]. Subsequently, Schafgen et al. reported two individuals with de novo truncating variants in TCF20 who presented with intellectual disability (ID) and overgrowth [14]. In addition, pathogenic variants in TCF20 have also been observed in two large cohort studies with cognitive phenotypes of ID and developmental delay (DD) [15, 16]. These isolated studies clearly support a role for TCF20 as a disease gene. However, a systematic study of patients with TCF20 pathogenic variant alleles from a cohort with diverse clinical phenotypes is warranted in order to establish a syndromic view of the phenotypic and molecular mutational spectrum associated with a TCF20 allelic series.

Interestingly, TCF20 shares substantial homology with a well-established Mendelian disease gene, RAI1, which is located in human chromosome 17p11.2 (MIM 607642). LoF mutations or deletions of RAI1 are the cause of Smith–Magenis syndrome (SMS; MIM 182290), a complex disorder characterized by ID, sleep disturbance, multiple congenital anomalies, obesity, and neurobehavioral problems [17–21], whereas duplications of RAI1 are associated with a developmental disorder characterized by hypotonia, failure to thrive, ID, ASD, and congenital anomalies [22, 23], designated Potocki–Lupski syndrome (PTLS; MIM 610883). Recent studies suggested that TCF20 and RAI1 might derive from an ancestral gene duplication event during the early history of vertebrates [9]. Therefore, it is reasonable to hypothesize that, as paralogous genes, mutations in TCF20 may cause human disease by biological perturbations and molecular mechanisms analogous to those operative in RAI1-mediated SMS/PTLS.

In this study, we describe the identification of TCF20 pathogenic variations by either clinical exome sequencing (ES) or clinical chromosomal microarray analysis (CMA) from clinically ascertained subjects consisting of cohorts of patients presenting with neurodevelopmental disorders as the major phenotype as well as with various other suspected genetic disorders. We report the clinical and molecular characterization of 28 subjects with TCF20 de novo or inherited pathogenic single nucleotide variants/indels (SNV/indels) and 4 subjects with interstitial deletions involving TCF20. These subjects present with a core phenotype of DD/ID, dysmorphic facial features, congenital hypotonia, and variable neurological disturbances including ataxia, seizures, and movement disorders; some patients presented features including sleep issues resembling those observed in SMS. Additionally, we report the molecular findings of 10 anonymized subjects with pathogenic TCF20 SNVs or deletion/duplication copy-number variants (CNVs). We demonstrate that ascertainment of patients from clinical cohorts driven by molecular diagnostic findings (TCF20 LoF variants) delineates the phenotypic spectrum of a potentially novel syndromic disorder.

We report 32 patients and 4 affected carrier parents with likely damaging pathogenic variants in TCF20. Phenotypic analysis of our patients, together with a literature review of previously reported patients, highlights shared core syndromic features of individuals with TCF20-associated neurodevelopmental disorder (TAND). Previous reports have collectively associated deleterious variants in TCF20 with ID, DD, ASD, macrocephaly, and overgrowth [6, 14–16] (Tables 1 and 2). The majority of the individuals in our cohort displayed an overlapping phenotype characterized by congenital hypotonia, motor delay, ID/ASD with moderate to severe language disorder, and variable dysmorphic facial features with additional neurological findings (Tables 1 and 2 and Fig. 2). We observe in our cohort that it is possible to have TCF20 deleterious variants transmitting across generations in familial cases (subjects #1, #5, and #7 and the twin brothers #27 and #28; Table 1, Additional file 1: Clinical information). Our parent carriers presented with an apparently milder phenotype; the mother of subject #1 showed mild dysmorphic facial features; the mother of subject #5 had features including ID, prominent forehead, tented upper lip, and short nose.

It is intriguing that TCF20 contains regions of strong sequence and structural similarity to RAI1 (Additional file 1: Figure S1) [22, 38–41]. RAI1 encodes a nuclear chromatin-binding multidomain protein with conserved domains found in many chromatin-associated proteins, including a polyglutamine and two polyserine tracts, a bipartite nuclear localization signal, and a zinc-finger-like plant homeodomain (PHD) (Additional file 1: Figure S1) [39]. A previous phylogenetic study of TCF20 and RAI1 suggested that a gene duplication event may have taken place early in vertebrate evolution, just after branching from insects, giving rise to TCF20 from RAI1, this latter representing the ancestral gene [9]. The two proteins share organization of several domains such as N-terminal transactivation domain, nuclear localization signals (NLS), and PHD/ADD at their C-terminus (Additional file 1: Figure S1) [9]. The PHD/ADD domain associates with nucleosomes in a histone tail-dependent manner and has an important role in chromatin dynamics and transcriptional control [42]. Here, we report that some patients with TCF20 mutations may present phenotypic features reminiscent of SMS such as craniofacial abnormalities which include brachycephaly, tented upper lips, midface hypoplasia, neurological disturbance (seizure, ataxia, abnormal gait), failure to thrive, food-seeking behaviors, and sleep disturbance.

To our knowledge, ataxia, hypertonia, food-seeking behavior, sleep disturbance, and facial gestalt reminiscent of SMS have not been previously reported in association with TCF20 pathogenic variants and represent a further refinement of TAND. Interestingly, subject #17 who presented features reminiscent of SMS harbors a missense variant c.5129A>G (p.Lys1710Arg) in the F-box/GATA-1-like finger motif part of the PHD/ADD domain in TCF20. The PHD/ADD domain that maps between amino acid positions 1690–1930 of TCF20 is highly conserved in RAI1 and confers the ability to bind the nucleosome and function as a “histone-reader” (HR) [8, 9]. Interestingly, mutations occurring in the region of GATA-1-like finger of RAI1 (p.Asp1885Asn and p.Ser1808Asn), in close proximity to the corresponding region of TCF20 where p.Lys1710 lies, are also associated with SMS [38, 39, 43].

Postnatal overgrowth has been previously reported in two patients with TCF20 defects [14]. We observe overgrowth, obesity, or tall stature in nine of the patients from our cohort. Interestingly, eight of these nine patients fall into an older age group (> 9.5 years old), representing 73% (8/11) of the patients older than 9.5 years old from our cohort; in the age group younger than 9.5 years old, only 6.7% (1/15) of them presented overgrowth. Further longitudinal clinical studies are warranted to dissect the etiologies of overgrowth, obesity, and tall stature, and to investigate whether these growth accelerations are age-dependent.

Of note, a subset of patients reported herein have sleep disturbance (38%, n = 12/32), hyperactivity (28%, n = 9/32), obsessive–compulsive traits (9%, n = 3/32), anxiety (6%, n = 2/32), and food-seeking behavior/early obesity (16%, n = 5/32) (Table 2), which could ultimately be attributed to circadian rhythm alterations as seen in SMS and PTLS [22, 38, 39]. Receptors for the steroid hormones estrogen (ER) and androgen (AR) have an emerging role in circadian rhythms and other metabolic function regulation in the suprachiasmatic nuclei in vertebrates through alteration of brain-derived neurotropic factor (BNDF) expression in animal models [44–47]. Interestingly, Bdnf is also downregulated in the hypothalamus of Rai1+/− mice, which are hyperphagic, have impaired satiety, develop obesity, and consume more food during light phase [48–50]. Since TCF20 has also been implicated in the regulation of ER- and AR-mediated transcriptional activity [10, 11, 51], we speculate that TCF20 might play a role in the regulation of circadian rhythms through steroid hormone modulation and disruption of its activity could lead to the phenotype observed in a subset of our patients.

Besides patient #17, all other patients carry either deletion or truncating variants occurring before the last exon of TCF20 that are predicted to be loss-of-function either through presumably NMD or by truncating essential domains of the TCF20 protein (Fig. 1). The frameshifting mutations from patients #27 and #28 are expected to result in a premature termination codon beyond the boundary of NMD, therefore rendering the mutant protein immune to NMD [37]. Future studies are warranted to delineate the exact correlation between genotype and phenotype in light of the potential escape from NMD and the potential pathway overlapping and interaction between TCF20 and RAI1 in the determination of the phenotype. It has been shown that around 75% of mRNA transcripts that are predicted to undergo NMD escape destruction and that the nonsense codon-harboring mRNA may be expressed at similar levels to wild type [52]. Therefore, alternative to NMD, we can speculate that, for instance, the truncating mutations that occur earlier in the gene before the first NLS (amino acid position 1254–1268) (Fig. 1, Additional file 1: Figure S1) in subjects #1 to #12 may determine loss-of-function of TCF20 due to either decreased level of protein in the nucleus with consequent cytoplasmic accumulation and/or to the absence of key functional C-terminal domains including PHD/ADD domains and/or DBD, AT-hook, NLS2, and NLS3, these latter representing unique motifs not conserved between TCF20 and RAI1 (Fig. 1, Additional file 1: Figure S1). It has been previously shown that the frameshift mutation c.3518delA (p.Lys1173Argfs*5) in TCF20 in one patient with ASD produces a stable mRNA that escapes NMD [6]. Data from our RNA studies corroborates this observation that TCF20 alleles with premature termination codon mutations may in general escape NMD. However, it should also be noted that NMD and mRNA turn over may be tissue specific and the current tissue tested is limited to blood. Based on this hypothesis, the position of amino acid truncation, for example, within the NLS or DNA-binding domain, may contribute to the prediction of genotype–phenotype correlation. The truncated TCF20 protein may retain partial function, representing hypomorphic alleles, or act in a dominant-negative manner sequestering transcription factors and co-factors in the absence of transcriptional modulation. Another possibility is that, due to the similarity between RAI1 and TCF20, mutated products of TCF20 could interfere with RAI1 pathways through the aforementioned mechanisms. Due to the complexity of the protein regulation and the variety of functional domains present in TCF20 (Additional file 1: Figure S1) that are not fully characterized, further studies are needed to refine the genotype–phenotype correlation.

Finally, although disorders associated with 22q13.2 deletions (encompassing TCF20) share similar features with Phelan–McDermid syndrome caused by deletion of SHANK3, our study provides evidence for the hypothesis that the major phenotypes observed in the former disorder are likely caused by direct consequence of TCF20 defects. Phenotypes specific for TCF20, such as sleep disturbances and movement disorders, may help clinically distinguish the 22q13.2 deletions from the 22q13.3 deletions (SHANK3). It is tempting to hypothesize that dosage gain of TCF20 may also be disease causing, given the similar observation at the 17p11.2 locus, where copy number gain of RAI1 was found to cause PTLS, potentially presenting mirror trait endophenotypes in comparison to SMS (e.g., underweight versus overweight) [53, 54]. This hypothesis predicts that TCF20 duplications are expected to cause similar neurodevelopmental defects as observed in the deletions, which is supported by the observation of TCF20 duplications from anonymized individuals with neurodevelopmental disorders, some of which are de novo (Fig. 2 and Additional file 1: Figure S1); additionally, one may speculate that specific phenotypes caused by TCF20 duplication may present mirror trait compared to those associated with the deletions, such as underweight versus overweight and schizophrenia spectrum disorders versus autism spectrum disorders. Further work is warranted to investigate the consequence of dosage gain of TCF20 in human disease.

Our findings confirm the causative role of TCF20 in syndromic ID, broaden the spectrum of TCF20 mutations recently reported, begin to establish an allelic series at this locus, and may help to understand the molecular basis of this new TAND syndrome. We also observe some patients with pathogenic variants in TCF20 presenting phenotypes reminiscent of SMS, suggesting potential common downstream targets of both TCF20 and RAI1. We suggest without molecular testing that it is challenging for a TAND diagnosis to be clinically reached purely based on the phenotypes observed in most patients. This underlines the importance of clinical reverse genetics for patients presenting with developmental delay and minor dysmorphic features, where positioning genotype-driven analysis (ES, CMA, or a combination of both) early in the “diagnostic odyssey” could improve the molecular diagnostic outcome and facilitate appropriate clinical management including recurrence risk counseling [55].