Haploinsufficiency of TCF4 Causes Syndromal Mental Retardation with Intermittent Hyperventilation (Pitt-Hopkins Syndrome)

In 1978, Pitt and Hopkins described two patients with sporadic “mental retardation, wide mouth and intermittent overbreathing.”¹ Since then, only four other sporadic cases with a similar phenotype and one sib pair with possible Pitt-Hopkins syndrome (PHS) have been published, but there is no MIM entry for this entity.^2–5 Breathing abnormalities in these published patients with PHS appeared in midchildhood, were present only when they were awake, and consisted of abrupt paroxysms of tachypnea followed by breath holding and even overt cyanosis. Other common findings were epilepsy with severe grand mal seizures, short stature, microcephaly, severe motor and mental retardation, and minor brain abnormalities such as cerebellar and vermis hypoplasia, hypoplasia of the corpus callosum, small hippocampus, and bulging caudate nuclei. Facial features were characterized by heavy supraorbital regions, a broad and beaked nose with a high bridge and flaring nostrils, a wide mouth, broad palate, and a bow-shaped upper lip.³

Since extensive metabolic studies and conventional karyotyping did not reveal any clues regarding the etiology, we performed molecular karyotyping⁶ using GeneChip Human Mapping 100K SNP arrays (Affymetrix) in the two sporadic cases published by Peippo et al.³ This work was performed as part of our research study addressing the genetics of mental retardation, which was approved by the Research Ethics Committee of the Medical Faculty of the University of Erlangen-Nuremberg. For molecular karyotyping, DNA samples were hybridized to GeneChip Human Mapping 50K Xba240 and Hind240 arrays, and images were obtained using an Affymetrix GeneChip Scanner 3000. Raw data were analyzed with the Affymetrix copy-number analysis tool (CNAT 2.0.0.9), with 0.5-Mb sliding windows for the genomic smoothing algorithm (GSA). Copy-number values calculated by the CNAT were filtered for clusters of SNPs with high GSA P values with use of a self-programmed software tool that we named “CNVFinder” (J. Hoyer, A. Dreweke, C. Becker, I. Göhring, C. Thiel, M. M. Peippo, R. Rauch, M. Hofbeck, U. Trautmann, C. Zweier, M. Zenker, U. Hüffmeier, C. Kraus, A. Ekici, F. Rüschendorf, P. Nürnberg, A. Reis, and A. Rauch, unpublished material). In one of the two patients analyzed, molecular karyotyping revealed a 1.2-Mb deletion on 18q21.2 (fig. 1A). The deletion was confirmed by FISH analysis with 18q21.2 BACs RP11-99A1 (RAB27B; 50.5–50.7 Mb), RP11-839G9 (CCDC68; 50.7–50.9 Mb), and RP11-7L24 (TCF4 [GenBank accession number NM_003199.1]; 51.1–51.24 Mb), all of which lacked the specific signal on one chromosome 18 homologue (GenBank accession number NT_025028.13) (fig. 1B). All three BACs gave normal FISH results in both parents, thus confirming de novo origin of the deletion in the patient. This deletion contained three known genes: RAB27B (a member of the RAS oncogene family), CCDC68 (coiled-coil domain containing 68), and TCF4 (transcription factor 4). Since, according to the UCSC Genome Browser database, only TCF4 is highly expressed in fetal and adult brain, we considered it the most likely candidate gene for the PHS phenotype. Sequence analysis of coding exons 2–19 and intronic flanking regions of TCF4 in the second patient published by Peippo et al.³ revealed the missense mutation R576/580W within exon 18, which codes for the helix-loop-helix (HLH) domain of TCF4. Sequencing was performed bidirectionally on an ABI 3730 capillary sequencer (Applied Biosystems) (detailed conditions and primer sequences are available on request). De novo origin of the missense mutation was proven by its exclusion in both parents. Paternity was verified, and mistake of probes was excluded by genotyping 14 polymorphic microsatellite markers in the child and both parents (PowerPlex 16 System [Promega]).

Because of phenotypic overlap with the Mowat-Wilson syndrome (MIM 235730), both patients had been tested for mutations in the ZFHX1B gene before this study.^3,7 We therefore screened 87 patients for TCF4 mutations in whom ZFHX1B testing had revealed normal results. None of these patients with mental retardation and variable features of the Mowat-Wilson syndrome spectrum, including constipation and Hirschsprung disease (HSCR [MIM 142623]), showed a TCF4 mutation.

We then sequenced 29 further patients with a more specific phenotypic overlap with PHS—that is, with at least two of the following features: severe mental retardation, breathing anomalies, and PHS-like facial dysmorphism. These patients also included a sib pair and a sporadic case formerly published as having PHS or possible PHS.^2,3,5 The original cases published by Pitt and Hopkins¹ were not available, because one patient died and the other was lost to follow-up (D. Pitt, personal communication). The case published by Singh⁴ could not be tracked either. TCF4 mutational analysis revealed three heterozygous stop mutations and a splice-site mutation in four of the unpublished patients (fig. 2 and table 1). De novo origin was proven in two patients (3 and 4), whereas parents of patients 5 and 6 were not available for testing. We therefore excluded both the R385X and IVS9-1G→C mutations of the latter patients in a total of 180 healthy European control individuals.

Since most of our patients have a deletion or stop or splice-site mutations, haploinsufficiency of TCF4 is likely to be causative of PHS. The only observed missense mutation affects an evolutionarily conserved amino acid and is located within the basic HLH (bHLH) domain of TCF4, thus likely impairing the binding capacity of the only functional domain known so far. TCF4 (also called “ITF2,” “E2-2,” and “SEF2”) consists of 20 exons (exons 1 and 20 are noncoding), spans 360 kb, and encodes at least two isoforms of the transcription factor 4 protein, differing in the presence of 4 aa (RSRS) 17 residues before the HLH domain. TCF4 belongs to the class A subfamily of bHLH transcriptional regulators—also called “E proteins,” since their basic domain binds to the E-box motifs 5′-ACANNTGT-3′ or 5′-CCANNTGG-3′.⁸ E proteins are characterized by a broad expression pattern and the ability to form both homo- and heterodimers with other classes of HLH proteins that are tissue specific or lack the basic DNA-binding domain.^9,10

Homozygous Tcf4 deletions in mice lead to early lethality of unknown reason and a slight decrease in pro-B cell numbers.¹⁰ In contrast to the apparently normal single heterozgyous Tcf4-knockout mice, transheterozygous knockout combinations of any two of the E proteins Tcf4, E2a, and Heb generate significantly reduced numbers of pro-B cells, and mice conditionally mutated in Tcf4 show a partial block in both B and T lymphocyte development.^9,10 Accordingly, none of our patients with heterozygous TCF4 mutations showed clinical evidence of immunodeficiency, and lymphocyte and Ig counts in patient 3 showed normal results. Nevertheless, patient 6 developed a Hodgkin lymphoma at age 29 years, which might indicate some kind of regulatory influence of TCF4 on lymphocyte growth. The diversified phenotypes observed with E-protein deficiencies are consistent with the idea that E proteins are involved in dimeric interactions with many different tissue-specific HLH proteins.¹⁰ Ascl1, the mouse homologue of one of these tissue-specific proteins, is highly expressed in specific regions of the developing CNS and in sympathetic and enteric precursor cells, and Ascl1-null mice die at birth.¹¹ ASCL1 was shown to form complexes with TCF4 that have the ability to bind an E box.¹¹ The interaction between TCF4 and ASCL1 is an interesting observation, since mutations in ASCL1 have been shown to impair the noradrenergic neuronal development in brain stem, causing some cases of congenital central hypoventilation syndrome (CCHS, or Ondine curse [MIM 209880]).¹² In Ascl1-knockout mice, an impaired c-RET expression in brain-stem noradrenergic neurons and an increased baseline breathing frequency were reported.¹³ The major gene that causes CCHS, PHOX2B, also belongs to the RET-signaling pathway, which, in mice, involves the sequential expression of the Ascl1, Phox, Ret, and TH genes that are responsible for the development of all transient or permanent noradrenergic derivatives.¹²

To investigate this interaction between TCF4 mutants and ASCL1, we established a transcriptional reporter assay using a luciferase reporter construct with a herpes simplex thymidine kinase promoter, either without binding sites (tkGL2) or with four E boxes (4xEtkGL2) located within the pTα enhancer, which interacts with several E proteins.¹⁴ JEG-3 cells (derived from human choriocarcinoma, American-type culture collection cell line HTB-36) were transiently cotransfected with a cytomegalovirus (CMV)–expression vector containing ASCL1, TCF4, its splice variant TCF4⁺ (including amino acid RSRS), either alone or in combination, and three different mutants of TCF4 and TCF4⁺ in combination with ASCL1 (fig. 3). Results were normalized, for transfection efficiency, to a cotransfected renilla luciferase vector.

In accordance with the results observed by Persson et al.,¹¹ who used a transcriptional reporter construct containing four E boxes from the muscle creatine kinase enhancer, in our assay, TCF4 and TCF4⁺ alone did not increase the activation of the reporter construct but enhanced the observed activation by ASCL1 when cotransfected with the latter. In contrast, TCF4 and TCF4⁺ mutants containing the mutations p.G232fsX256, R385X, or R576/580W did not enhance the activation by ASCL1 (fig. 3). Therefore, breathing anomalies in patients with PHS may also be caused by impaired noradrenergic neuronal development through defective TCF4 interaction with the ASCL1-PHOX-RET pathway. This interaction might also explain the occurrence of HSCR and constipation in patients 1, 2, and 5, since RET is the major gene for isolated HSCR¹⁵ and since patients with CCHS show an increased incidence of HSCR (20%).¹⁶ However, no obvious defect in the sympathetic nervous system in Tcf4-knockout mice was observed that could be attributed to a functional interaction with Ascl1.¹¹ In contrast to patients with CCHS, who are intellectually not impaired, patients with PHS are severely mentally retarded, which may indicate TCF4 involvement in other pathways important for brain development and function corresponding to its high expression in brain.¹⁷

All six patients with TCF4 mutations showed severe mental retardation and striking facial resemblance, at least to patient 1 initially reported by Pitt and Hopkins,¹ consisting of deep-set eyes; broad and beaked nasal bridge with down-turned, pointed nasal tip and flaring nostrils; wide mouth with widely spaced teeth, Cupid-bowed upper lip, and everted lower lip; and mildly cup-shaped, fleshy ears (fig. 4). Further findings were variable, including breathing abnormalities, which were not yet obvious in patient 3 at age 8 years (table 1). Other common signs were magnetic resonance imaging (MRI) anomalies such as hypoplastic corpus callosum and bulging caudate nuclei, happy disposition or unmotivated laughter episodes, muscular hypotonia, severe constipation or HSCR, single palmar creases, and supernumerary digital flexion creases. The manifestation of a lymphoma in one of the oldest patients may be attributed to the role of TCF4 in lymphocyte development,^9,10 but further observations and TCF4 studies in lymphoma tissues are necessary to address this question.

Since most of the patients in whom TCF4 mutations were identified had previous testing for Angelman syndrome (MIM 105830) because of facial and behavioral resemblance and for Rett syndrome (MIM 312750) because of late-onset ventilation anomalies and severe mental retardation, PHS might be an important differential diagnosis for these disorders. Interestingly, one patient with severe mental retardation and attacks of deep sighing and hyperventilation who carries an interstitial deletion (18)(q21.1q22.3) was reported as a case of “atypical Rett syndrome.”¹⁸ In contrast, Joubert syndrome (MIM 213300, 608091, 609583, 608629, 610688, and 610188) was never considered in any of these patients, because its characteristic intermittent hyperventilation-apnea anomaly manifests in the newborn. Although most of the patients showed more or less overlapping features, we were unable to demonstrate a mutation in the remaining 24 patients, including the sporadic case published by Van Balkom et al.⁵ and the sib pair described by Orrico et al.² Although larger gene deletions were excluded by the presence of heterozygous SNPs in all but 2 of the 24 patients, we cannot exclude atypical intronic mutations or single-exon deletions in the 24 mutation-negative patients. Nevertheless, the large number of mutation-negative patients with more or less similar phenotype might indicate the involvement of further genes that possibly interact with TCF4.

Our study shows that molecular karyotyping^6,19,20 (genomewide copy-number profiling) is able not only to disclose novel microdeletion syndromes^21–23—as well as the underlying gene defect in well-known disorders, as recently shown for the autosomal dominant CHARGE (coloboma, heart anomaly, choanal atresia, retardation, genital and ear anomalies) syndrome (MIM 214800)²⁴ and the autosomal recessive Peters-Plus syndrome (MIM 261540)²⁵—but also to resolve the etiology in very rarely reported phenotypes. However, our findings suggest that PHS is widely underdiagnosed, just as the now clinically recognizable Mowat-Wilson syndrome was until the identification of the underlying gene defect, in 2001.^7,26,27 Notably, PHS is, to our knowledge, the first constitutive phenotype in which the etiology was identified by molecular karyotyping with use of genomewide SNP arrays.