Haploinsufficiency of TCF4 causes syndromal mental retardation with intermittent hyperventilation (Pitt-Hopkins syndrome)

Abstract

Pitt-Hopkins syndrome is a rarely reported syndrome of so-far-unknown etiology characterized by mental retardation, wide mouth, and intermittent hyperventilation. By molecular karyotyping with GeneChip Human Mapping 100K SNP arrays, we detected a 1.2-Mb deletion on 18q21.2 in one patient. Sequencing of the TCF4 transcription factor gene, which is contained in the deletion region, in 30 patients with significant phenotypic overlap revealed heterozygous stop, splice, and missense mutations in five further patients with severe mental retardation and remarkable facial resemblance. Thus, we establish the Pitt-Hopkins syndrome as a distinct but probably heterogeneous entity caused by autosomal dominant de novo mutations in TCF4. Because of its phenotypic overlap, Pitt-Hopkins syndrome evolves as an important differential diagnosis to Angelman and Rett syndromes. Both null and missense mutations impaired the interaction of TCF4 with ASCL1 from the PHOX-RET pathway in transactivating an E box-containing reporter construct; therefore, hyperventilation and Hirschsprung disease in patients with Pitt-Hopkins syndrome might be explained by altered development of noradrenergic derivatives.

Figure 1.

Results of molecular karyotyping with Affymetrix GeneChip Human Mapping 100K SNP array in patient 2. A, Plot of ±log₁₀ GSA P values for SNPs covering chromosome 18, with use of a Gnuplot program. The 1.2-Mb deletion in 18q21.2 is visible through a cluster of SNPs with GSA P values <−2.5. The 63 SNPs indicating a deletion are flanked by SNP_A-1695165 (rs4800947; 50,552,638 Mb) and SNP_A-1724163 (rs784395; 51,742,365 Mb). Genotypes of individual SNPs are indicated by colored bars at the bottom (magenta=homozygous; blue=heterozygous; brown=no call). Note the magenta stretch of homozygosity corresponding to the deleted region. B, Representative results of two-color FISH analyses, with the RP11-7L24 probe labeled with Cy3 (pink) in combination with a FluoroX-labeled (green) subtelomeric 18p control probe, for patient 2 and his parents. Whereas the RP11-7L24 probe is lacking on one chromosome 18 homologue in the patient, it is present on both homologues in the parents, demonstrating de novo origin of the deletion in patient 2.

Figure 2.

Schematic drawing of the 1.2-Mb deletion region in patient 2 and location and electropherograms of TCF4 mutations in patients 1, 3, 4, 5, and 6 within a schematic drawing of exon-intron structure of TCF4. Noncoding exons are light gray, and the exon coding for the functional domain is black. Blue bars representing BAC clones and blue squares representing SNPs indicate deletion of respective probes, whereas nondeleted SNPs are depicted as black squares.

Figure 3.

Transcriptional reporter assay showing impaired interaction of TCF4/TCF4⁺ mutants with ASCL1. JEG-3 cells were transiently transfected with 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. Cotransfection was performed with an empty CMV-expression vector (white) or CMV-expression vectors containing the complete cDNA of either ASCL1 (red), TCF4 (pale yellow), its splice variant TCF4⁺ (including amino acid RSRS) (bright yellow), as well as wild-type or three different mutants of TCF4 and TCF4⁺, transfected in combination with ASCL1 (different shades of orange). Results were normalized for transfection efficiency to a cotransfected renilla luciferase vector and were expressed as mean values with SD of three independent transfections. Probably because of endogenous E proteins, cells transfected with the empty CMV vector alone already showed a slight transactivation of the luciferase vector containing the four E boxes (4xEtkGL2), in comparison with the reporter vector without E boxes (tkGL2). 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 mutation p.G232fsX256, R385X, or R576/580W did not enhance the activation by ASCL1. Differences in activation levels showed significant P values obtained by the Student t test.

Figure 4.

Facial phenotype of patients with TCF4 mutations. A–C, Patient 1 at ages 6 mo (A), 18 mo (B), and 14 years (C). D and H, Patient 6 at age 29 years. E–G, Patient 2 at ages 6 mo (E and F) and 11 years (G). I–K, Patient 3 at ages 3 years (I), 6 years (J), and 8.75 years (K). L and M, Patient 4 at age 12.5 years. Note deep-set eyes; broad and beaked nasal bridge with down-turned, pointed nasal tip and flaring nostrils; wide mouth with widely spaced teeth, and Cupid-bowed upper lip and everted lower lip; mildly cup-shaped, fleshy ears; as well as increased coarsening of facial features with age.