The recurrent TCF4 missense variant p.(Arg389Cys) causes a neurodevelopmental disorder overlapping with but not typical for Pitt‐Hopkins syndrome

Abstract TCF4 haploinsufficiency by deletions, truncating variants or loss‐of‐function missense variants within the DNA‐binding and protein interacting bHLH domain causes Pitt‐Hopkins syndrome (PTHS). This neurodevelopmental disorder (NDD) is characterized by severe intellectual disability (ID), epilepsy, hyperbreathing and a typical facial gestalt. Only few aberrations of the N‐terminus of TCF4 were associated with milder or atypical phenotypes. By personal communication and searching databases we assembled six cases with the novel, recurrent, de novo missense variant c.1165C > T, p.(Arg389Cys) in TCF4. This variant was identified by diagnostic exome or panel sequencing and is located upstream of the bHLH domain. All six individuals presented with moderate to severe ID with language impairment. Microcephaly occurred in two individuals, epilepsy only in one, and no breathing anomalies or myopia were reported. Facial gestalt showed some aspects of PTHS but was rather non‐specific in most individuals. Interestingly, the variant is located within the AD2 activation domain next to a highly conserved coactivator‐recruitment motif and might alter interaction with coactivator proteins independently from the bHLH domain. Our findings of a recurrent missense variant outside the bHLH domain in six individuals with an ID phenotype overlapping with but not typical for PTHS delineate a novel genotype–phenotype correlation for TCF4‐related NDDs.

most individuals. Interestingly, the variant is located within the AD2 activation domain next to a highly conserved coactivator-recruitment motif and might alter interaction with coactivator proteins independently from the bHLH domain. Our findings of a recurrent missense variant outside the bHLH domain in six individuals with an ID phenotype overlapping with but not typical for PTHS delineate a novel genotype-phenotype correlation for TCF4-related NDDs. to E-box consensus motifs, was identified as the underlying molecular cause. 2,3 PTHS is characterized by severe intellectual disability (ID), lack of or significantly impaired speech development, behavioral issues, seizures, constipation, early-onset myopia as well as a recognizable facial gestalt. 4 The latter is characterized by a prominent nasal bridge, a beaked nose, prominence of the lower face and a wide mouth with a Cupid's bow shaped upper lip. 4,5 Additionally, a narrow forehead, thin lateral eyebrows, flared nasal alae, full cheeks and thickened/ overfolded helices were delineated as distinctive. 6 The majority of PTHS related aberrations affect exons 9-19 of TCF4 (NM_003199.2; NM_001083962.2; 20 exons) and comprise large deletions, intragenic deletions, truncating and few elongating variants. About 20% of causative variants are missense variants located in the highly conserved bHLH domain, encoded by exon 18 (overview in Zollino et al. 6 ).
Of note, several (likely) pathogenic TCF4 variants have been associated with milder or non-specific ID. While variants within the very N-terminal exons 1-4 were found in individuals with mild ID, 7 in some instances even inherited within a family, 8,9 loss-of-function variants in exons 7 and 8 seem to be associated with severe ID with or without typical features of PTHS. 7,10 We now report on six individuals harboring a recurrent missense variant (confirmed to be de novo in five individuals) upstream of the bHLH domain and presenting with rather non-specific moderate to severe ID without or with only few facial aspects of PTHS.

| METHODS AND RESULTS
By personal communication among colleagues, being contacted by one of the families, and by searching ClinVar, 11 Varsome, 12 and Decipher, 13  Genetic analysis was performed by either diagnostic trio exome sequencing in three cases or sequencing clinical exome or panels of 56 or nine ID genes, respectively, in the others ( Table 1). The p.
(Arg389Cys) variant is not reported in gnomAD. 14 It occurs three times in ClinVar 11 with conflicting interpretation of pathogenicity (2Â likely pathogenic, 1Â with uncertain significance). One of the ClinVar cases is included in this study (SCV002011164.1). The variant affects a highly conserved amino acid and is predicted to be pathogenic by multiple in silico predictors (e.g., SIFT, Polyphen-2, CADD).
Clinical details of the six affected individuals are summarized in Table 1. All had moderate to severe ID with lack of or severely   As reflected by three clinical scoring systems, the most distinctive clinical aspect of PTHS is the recognizable facial gestalt. 6,15,16 Whereas one of the individuals clearly resembled PTHS, this was less or not apparent in the other individuals. While single or few dysmorphism such as overfolded helices and/or a beaked nose were noted in most of them and do overlap with PTHS, the overall facial gestalt would, in our opinion, not allow a "facial diagnosis" of PTHS.
Utilizing the most recently published clinical scoring system, 6 none of the six individuals would reach a score allowing clinical diagnosis of PTHS and only one would reach a score indicating possible PTHS (Table 1) preference. 18 The variant c.759C > G, p.(Ser253Arg) in exon 10 was reported in a male individual with Rett-like features but without clinical photographs. 19 The same variant is contained three times in ClinVar as likely pathogenic and was shown to result in slightly reduced DNA binding and transactivation. 20