Various haploinsufficiency mechanisms in Pitt-Hopkins syndrome

Abstract

Pitt-Hopkins syndrome is a rare neurodevelopment disorder caused by haploinsufficiency of the transcription factor 4 (TCF4). The main clinical symptoms of Pitt-Hopkins syndrome are severe development delay, intellectual disability, characteristic facial phenotype, and breathing abnormalities, including episodic hyperventilation. Different pathogenic variants can lead to Pitt-Hopkins syndrome. The most common are large deletions at 18q21 encompassing the TCF4 gene and frameshifting/nonsense single nucleotide variants. However, variants in noncoding regions can also lead to Pitt-Hopkins syndrome by disrupting the normal pre-mRNA splicing machinery. Here we describe three patients with Pitt-Hopkins syndrome caused by a large deletion in chromosome 18, a nonsense variant, and a novel variant located in intron 11 of TCF4 c.922+5G > A. Using RT-PCR analysis and minigene splicing assay we showed that this intronic variant leads to exon 11 skipping resulting in a formation of a premature stop codon. To our knowledge, this is the first functional annotation of a splicing variant in Pitt-Hopkins syndrome.

Introduction

Pitt-Hopkins syndrome (PTHS; MIM #610954) represents a rare neurological condition first described in two unrelated patients in 1978 (Pitt and Hopkins, 1978) with intellectual disability, wide mouth, and episodic hyperventilation. Since then the clinical picture of PTHS expanded and the most consistent symptoms are severe development delay, intellectual disability, most often with absent speech and different breathing abnormalities. Seizures, autism spectrum disorder symptoms, constipation, and myopia are also frequently present in patients with PTHS along with several dysmorphic facial features (Whalen et al., 2012). The molecular causes of PTHS are heterozygous pathogenic variants or large deletions in chromosome 18 including the TCF4 gene and the main mechanism is haploinsufficiency (Amiel et al., 2007; Zweier et al., 2007). A large proportion of intergenic causes of PTHS are recurrent pathogenic missense variants in exon 18 of the TCF4 gene located in the basic helix-loop-helix (bHLH) domain and disrupt the DNA binding ability of TCF4 (Whalen et al., 2012; Thaxton et al., 2018). The TCF4 gene encodes a basic helix-loop-helix E-protein that acts as a transcription factor with a broad expression pattern (Jung et al., 2018) and is crucial during early development of the brain (Forrest et al., 2014). The prevalence of PTHS is unknown. Some authors estimate it as 1:34000–1:41000 (Rosenfeld et al., 2009), however to date only about 500 patients were described worldwide (Orphanet Report Series -, 2020).

Here we describe three patients with PTHS with detailed clinical characterization and different molecular mechanisms of haploinsufficiency. For one of the patients harboring the intronic variant c.922+5G>A a functional analysis was performed to confirm the pathogenicity of the novel variant.