A genotypic and phenotypic analysis of four unrelated Chinese patients with Pitt–Hopkins syndrome

Abstract

Background

Pitt–Hopkins syndrome (PTHS) is a rare genetic condition caused by a mutation in the transcription Factor 4 (TCF4) gene and characterized by its unique clinical presentations. At present, there is an incomplete understanding of the possible TCF4 mutations and their downstream consequences, and no reliable treatment exists for patients with PTHS. Elucidating the variations in TCF4 occurring in PTHS could lead to new treatment ideas.

Case presentation

We described the clinical and genetic characteristics of four Chinese patients with PTHS. Genetic mutations related to central nervous system (CNS) disorders were identified via high-throughput sequencing. The patient’s mutations were subsequently confirmed with Sanger sequencing. Most patients had facial features typical of PTHS; however, Patient 2 demonstrated poor auricle shape. Each patient presented with differing levels of delayed development and intellectual impairment. The patients showed a splice site mutation in intron 15 of TCF4 (c.1452 + 3A > G), a frameshift mutation in exon 18 of TCF4 (c.1942delA), and two missense mutations in exon 19 of TCF4 (c. 2147C > T and c.2026A > G).

Conclusions

We discovered four new TCF4 mutations in Chinese children with PTHS. To our knowledge, the c.2026A > G and c.1942delA mutations have not yet been reported. The detection of these mutations can help accurately diagnose PTHS early, especially in patients whose clinical symptoms are not obvious. Exploring the genotype and phenotype of individuals with PTHS, will enrich our understanding and guide further research into the role of TCH4.

Introduction

PTHS (OMIM #610,954) is rare neurodevelopmental disorder characterized by distinct facial features, developmental delays, intellectual impairments, and respiratory symptoms. It was first described in 1978 in two unrelated patients [1]. With an increasing number of cases reported, our understanding of the clinical presentation of PTHS has improved. For example, patients with PTHS also frequently present with seizures, autism spectrum disorder symptoms, constipation, and myopia [2]. Given these clinical characteristics, the symptoms of this disease resemble those of other neurodevelopmental disorders, including Angelman syndrome, Rett syndrome, Mowat–Wilson syndrome, and ATR-X syndrome. The frequency of this disease occurrence remains uncertain, with approximately 500 PTHS patients having been reported in the literature thus far [2].

The TCF4 (OMIM #602,272) gene is located at chromosomal region 18q21.2. This gene encodes a basic helix‒loop‒helix (bHLH) transcription factor that binds to cis-regulatory elements of target genes and regulates gene transcription [3]. TCF4 is critical for early brain development, neurogenesis, and ion transport, and deletions or variants of this gene cause PTHS [4, 5]. Misprints and large deletions of TCF4, including chromosomal translocations, intragenic deletions, truncations and likely gene disruptive or missense variants, are implicated in nervous system development. Over 140 different mutations in TCF4 have been documented in the literature [2]. The variety of TCF4 mutations and numerous affected downstream processes could explain the wide range of clinical presentations in patients with PTHS [6]. Although there is currently no effective treatment for PTHS, symptom managements is conducted similarly to the procedures recommended for the general population, as per the most recent international consensus [2].

Herein, we report four Chinese PTHS patients with molecularly confirmed mutations (2 novel and 2 known) in TCF4 and describe the corresponding clinical presentations in detail. Variable levels of intellectual disability and developmental delay were evident in these four patients with mutations. However, not all patients presented with typical facial features. TCF4 gene mutation detection may help diagnosis of PTHS early.

Materials and methods

The Ethics Review Committee of the Affiliated Hospital of Qingdao University approved this research. Consent for genetic testing and the release of related information was given by the patients' parents. Past medical data were collected from the four patients who were referred to Qingdao Women & Children's Hospital and were diagnosed with PTHS with molecular testing. Informed consent for the publication of the clinical data and genetic test results identified by whole exome sequencing (WES) was obtained. According to the most recent diagnostic guidelines for PTHS [2], a clinical diagnostic score was determined for every patient. A score of 9 or above represented a confirmed clinical diagnosis of PTHS. However, a score of 6 to 8 and abnormal facial characteristics, indicated possible PTHS with the need to obtain additional validation through molecular testing. Choosing a total of 1700 candidate genes linked to CNS disorders as our genes of interest, we utilized a specially designed gene panel to capture the coding regions of these selected genes, encompassing their exons and the boundaries between exons and introns. We used targeted next-generation sequencing technology to identify the pathogenic mutations associated with CNS disorders. We verified the recognized pathogenic mutations and noted polymorphisms by examining the Professional Human Gene Mutation Database (HGMD), the National Center for Biotechnology Information (NCBI) 1000 Genomes Browser, PubMed, and the single nucleotide polymorphism (SNP) database. Sanger sequencing confirmed the identified mutation. We compared the results with the TCF4 sequence from GenBank (NM_001243226).

Results

Patient 1

A Chinese female patient with no known family history of intellectual, neurological, or psychiatric ailments was evaluated at 16 months old. She was delivered through a cesarean section at the end of a complication-free pregnancy, and her neonatal phase was regular. However, she exhibited delayed motor milestone development; for example, she could only sit without support at 14 months, and at 16 months, she could not independently stand or climb. Her language development was severely impaired, with her vocabulary being composed of only a handful of words. She had a cheerful temperament and regularly exhibited stereotypic behaviors such as hand flapping and rocking. Minor dysmorphic features included a noticeably thin lateral eyebrow, wide nasal bridge and tip, flared nostrils, full cheeks, a wide mouth, a thickened helix, and a simian crease (Fig. 1A). Her limb muscle tension and patellar tendon reflex were low. She had never experienced any seizures, sleep disorders, or constipation. Her vision was normal. She had never developed Hirschsprung syndrome and had a high respiratory rate. Her palate was normal, and her head circumference was within the normal range. Her developmental quotient (DQ) was assessed via the 1986 Gesell developmental scale (GDS) [7]; the resulting GDS score was 55, indicating mild mental retardation. The following questionnaires were used to measure growth and development: the second edition of the Age and Steps Questionnaire of Social and Emotional Development (ASQ-SE-2) developed by Squires et al. [8] and the third edition of the Child Development Screening Test (ASQ-3) developed by Squires and Bricker [9]. The results were all subthreshold, suggesting developmental delay.

Fig. 1 [Images not available. See PDF.]

Facial phenotype of the 4 patients reported in the manuscript. A Patient 1, aged 16 months; B patient 2, aged 19 months; C patient 3, aged 38 months; D patient 4, 25 months

The patient's chromosomal microarray initially presented as normal. However, upon whole-exome sequencing, a diagnosis of PTHS was given. The patient had a splice-site mutation, c.1452 + 3A > G (Fig. 2A), which was notably absent in the TCF4 genes of the patient's parents. This variant is considered a de novo mutation and is likely to impair protein function. The pathogenicity of this variation has been reported [10]. Retrospective analysis of Patient 1’s phenotype and comparison with PTHS diagnostic criteria [2] yielded a score of 8 points (6 cardinal points and 2 supportive points).

Fig. 2 [Images not available. See PDF.]

Sanger sequencing of the detected isoforms. A The patient 1 with the c.1452 + 3A > G mutation in TCF4 gene. B The patient 2 with the c.2147C > T, p.Ala716Val mutation in TCF4 gene. C The patient 3 with the c.2026A > G, p.Asn676Asp mutation in TCF4 gene. D The patient 4 with the c.1942delA, p.Arg648GlyfsTer12 mutation in TCF4 gene

Patient 2

At the time of her most recent evaluation, Patient 2 was a 19-month-old girl from China. No family history of neurologic disorders was noted. At the developmental stage of 12 months, the patient was reported to be capable of sitting and babbling, but not of forming any distinct words. At her first evaluation at 19 months, she was unable to climb. She had motor learning impairments and no signs of language. Her vision had not yet been tested. She never experienced seizures, sleep disorders, or constipation. She had never developed Hirschsprung syndrome and had a high respiratory rate. Her palate was normal, and her head circumference was within the normal range.

She presented mildly dysmorphic facial features with poor auricle shape (Fig. 1B) and weak reactions to verbal commands. Her limb muscle tension and patellar tendon reflex were low. Magnetic resonance imaging (MRI) of the child’s brain revealed ventriculomegaly in the cerebral area and a blunt anterior horn. Her social ability was slightly impaired. Routine electroencephalography (EEG) was normal. She was diagnosed with autism spectrum disorder and severe intellectual disability.

WES revealed a missense mutation in exon 19 of TCF4, c.2147C > T (p.Ala716Val) (Fig. 2B), which has been previously reported [11].

The TCF4 gene of the parents did not contain a missense mutation, suggesting that the patient had a de novo mutation. Retrospective analysis of Patient 2’s phenotype and comparison with PTHS diagnostic criteria [3] yielded a score of 3 points (2 cardinal points and 1 supportive point).

Patient 3

The initial clinical examination of Patient 3 was conducted due to observed developmental delays when she was 3 years 2 months old. She was born via cesarean section at full term after a typical gestation period, but her parents were not involved. Her neonatal phase proceeded normally. Despite this, her motor development was delayed: she could hold her head at 3 months, roll at 6 months, sit at 8 months, and climb at 20 months. At the time of the initial evaluation, she was able to pull herself up to a standing position but lacked the ability to stand or walk without assistance. She had stiff hands and no sign language except being able to sign goodbye. She had problems with verbal memory and language development. She could express only single words, such as no, dad, mum, and ah, lacking meaningful or active language. The patient presented with the following facial features: a narrow forehead, wide nasal bridge, flared nostrils, full cheeks, and a cupid bowing of the upper lip (Fig. 1C). She had no response to verbal instructions and could not imitate. She showed knee extension when standing and forefoot pronation and trunk shaking when walking. Neurological examination revealed moderate hypotonia and hyporeflexia. She was developmentally delayed and had autism spectrum disorder. No instances of constipation or seizure-like activity were reported. Her sleep was uninterrupted. She had never developed Hirschsprung syndrome and had a high respiratory rate. Her palate was normal, and her head circumference was within the normal range. Her EEG results were normal with no epileptic activity, and cerebral MRI results were normal. Her social ability was severely impaired.

WES revealed a previously undescribed missense mutation in exon 19 of the TCF4 gene, c.2026A > G (p.Asn676Asp) (Fig. 2C). The presence of the variant in the proband was confirmed through Sanger sequencing, but it was not found in the patient’s parents or her brother, indicating that her variant was a de novo mutation. According to the ACMG guidelines [11], the variant was classified as class 4 (PS2, PM1, PM2, PP3, BP1). This variant leads to the amino acid deletion p. Asn676Asp, which is possibly pathogenic (SIFT: deleterious; Polyphen2: probably damaging; Mutation Taster: disease causing). Retrospective analysis of Patient 3’s phenotype and comparison with PTHS diagnostic criteria [2] yield a score of 7 points (6 cardinal points and 1 supportive point).

Patient 4

Patient 4 was an 8 months and 15 days old Chinese boy at the time of evaluation with no family history of intellectual disability or other neurological or psychiatric disorders. A cesarean section was performed at 37 weeks of gestation. His family reported that he was treated to address choking on his amniotic fluid at a local hospital for 12 days. The specific treatment and diagnosis are unknown. His birth weight was 3100 g. The infant experienced delayed early motor milestones—he was able to hold his head up only when he was 2.5 months old, and he could not roll, sit with hand support, perform abdominal climbs, stand alone or stand with support. His language development was severely impaired, with only a few words in his vocabulary. There were only a few words in the vocabulary. He was also generally developmentally delayed. The following minorly dysmorphic features were evident at the 2 years and 3 months: hypotelorism, a thin lateral eyebrow, a wide nasal bridge and tip, flared nostrils, a prominent midface, a cupid bow upper lip, and a thickened helix (Fig. 1D). He had never experienced any seizures, sleep disorders, constipation, or difficulties with vision. He had never developed Hirschsprung syndrome and had a high respiratory rate. His palate was normal and his head circumference was within the normal range. His EEG activity was normal without any epileptic activity. His Alberta Infant Motor Scale (AIMS) score was < 1st percentile, and the MS results of the Gesell test indicated a critical level.

WES revealed a previously undescribed variant of the TCF4 gene, c.1942delA p.Arg648GlyfsTer12 (Fig. 2D). The variation was not present in the TCF4 gene of the parents. The variant was classified as class 4 according to the American College of Clinical and Genomics [12]. This heterozygous nucleotide mutation results in a change in amino acid synthesis beginning with amino acid no.648 Arg, and in the altered 12th amino acid termination (p.Arg648GlyfsTer12). No reports on the pathogenicity of this variant have been published in the literature according to the HGMD Pro and PubMed reference databases. Retrospective analysis of Patient 4’s phenotype and comparison with PTHS diagnostic criteria [2] yielded a score of 7 points (6 cardinal points and 1 supportive point).

The detailed clinical information is summarized in Table 1.

Table 1. Detailed clinical information of the PTHS patients described in the paper according to HPO terms

HPO term	Signs	Patient 1	Patient 2	Patient 3	Patient 4
HP:0001511	Intrauterine growth retardation	−	−	−	−
HP:0000341	Narrow forehead	−	−	+	+
HP:0005338	Thin lateral eyebrown	+	−	−	+
HP:0000431/0000455	Wide nasal bridge/tip	+	−	+	+
HP:0000454	Flared nostrils	+	−	+	+
HP:0000293/0012371	Full cheeks/prominent midface	+	−	+	+
HP:0000154/0012471/ 0002263	Wide mouth/full lips/cupid bow upper lip	+	−	+	+
HP:0000391/0000396	Thickened helix/overfolded helix	+	+	+	+
HP: 0010864	Severe intellectual disability	+	+	+	+
HP:0001344	absent speech	+	+	+	+
HP:0009062	Infantile axial hypotonia	+	+	+	+
HP:0002194	Delayed gross motor development	+	+	+	+
HP:0004879	Intermittent hyperventilation	−	−	−	−
HP:0002019	Constipation	−	−	−	−
HP:0000545	Myopia	−	−	−	−
HP:0000486	Strabismus	−	−	−	−
HP:0000483	Astigmatism	−	−	−	−
HP:0000253	Microcephaly	−	−	−	−
HP:0001250	Seizures	−	−	−	−
HP:0002119	Wide ventricles	−	−	−	−
HP:0007370	Small corpus callosum	−	−	−	−
HP:0000954	Single transverse palmar crease	+	−	−	−
	Pathogenic variant
	PTHS score	8	3	7	7

PTHS score was calculated according to PTHS diagnostic criteria [3]

Discussion

The phenotype of patients with PTHS has a characteristic pattern; nevertheless, not all individuals with PTHS have typical clinical features or a variant in TCF4. Furthermore, owing to its relatively low prevalence, clinicians may have difficulties diagnosing this disorder. Another important challenge for diagnosis is that PTHS patients often have phenotypes similar to those of patients with other syndromes, such as Rett and Angelman syndrome [13]. Therefore, even though there are published diagnostic guidelines for PTHS [2], there is still a need for additional descriptions of novel clinical cases to increase our knowledge and understanding of this syndrome.

There is currently no effective treatment for individuals with PTHS. Nonetheless, progress has been made in the analysis of the cellular, molecular, physiological, and behavioral aspects of TCF4 in PTHS, with an emphasis on characterizing phenotypes and guiding the development of new treatment methods. In this study, three of these four patients had different de novo mutations in exons 18–19 of the TCF4 gene, and variable degrees of developmental delay and intellectual disability. In particular, Patient 2 who showed a missense mutation in exon 19, exhibited mild special facial features. Similarly, a milder clinical phenotype was reported in a Chinese female with PTHS caused by a missense mutation in exon 18 [6]. Previous studies have reported that impairment of different protein domains causes variable clinical presentation within PTHS and mutations in exons 9–19 typically cause PTHS [14]. Nonetheless, this conclusion does not hold true in all PTHS cases. We also found that Patient 2 and 3, with missense mutations in exon 19, presented with distinct clinical phenotypes. Related studies have shown that missense mutations that affect DNA binding directly or indirectly cause typical PTHS, whereas missense mutations that induce only subtle conformational changes lead to a milder clinical phenotype [15]. This may contribute to the phenotypic variability of PTHS patients. Studies on the downstream functions of TCF4 and the establishment of animal models suggest that neural sodium channel gene 10a (SCN10a) is a potential therapeutic target for PTHS [16]. Hence, comprehensively characterizing the genotype and phenotype of PTHS individuals, particularly those with recently identified mutations, will not only enrich our understanding of and motivate further research on TCH4 function but also present new ideas for PTHS treatments.

In our research, we identified four patients, each of whom presented with a unique de novo mutation in TCF4, and varying levels of developmental delay and intellectual disability. None of these patients could stand without assistance, with the Patients 1 and 2 displaying reduced muscle tension in their lower limbs and a diminished patellar tendon reflex. All patients had severe language delays. During the assessment period, all the subjects could babble but lacked any distinct words. Commonly employed functional assessments of adaptation and initial developmental skills, such as the DQ, ASQ-SE-2, and ASQ-3, modified Checklist for Autism in Toddlers, Alberta, and GESELL, were conducted. Our observations align with the majority of documented cases in which speech is typically absent, and significant motor delays are present [17–20].

In the identification and differentiation of PTHS from other syndromes, the principal identifying characteristic is the usual facial gestalt [2]. However, not all patients in this study exhibited presented with facial features typical of PTHS. Most participants in this study displayed these typical facial attributes; however, Patient 2 only showed an altered auricle shape. Notably, Patient 2 would not have been diagnoses with PTHS using the latest proposed international clinical diagnostic scores [2]. Hence, the underdiagnosis of PTHS is likely, particularly when the clinical phenotype and facial features are not very distinctive.

WES has proven a valuable tool in the characterization of underlying genetic defects in most rare diseases (RDs) [21]. WES can simultaneously detect genetic variants associated with most diseases. However, in addition to the focused variants suspected to be clinically relevant, the detected variants also contained other variants of uncertain significance. The possibility of false-positives cannot be completely ruled out. Therefore, further validation work is recommended for variants that are suspected to be clinically significant. All of the patients described here had available WES genetic testing results. The chromosomal microarray of Patient 1, who was suspected of having COVID-19 on the basis of the latest published diagnostic guidelines [2], was normal, and he was later diagnosed with PTHS by WES. Additionally, Patient 2, who would not have been recommended on the basis of the latest published diagnostic guidelines [2], was genetically tested for PTHS and diagnoses, thus indicating that the available diagnostic tools and approaches are still limited. Previous studies have shown that young patients are more likely to be underdiagnosed, partly because these patients are too young to show symptoms [13, 18]. We can further confirm that WES can improve the ability to accurately diagnose patients early, especially patients whose clinical symptoms are not obvious. In addition, the various haploinsufficiency mechanisms of Pitt–Hopkins syndrome [22] necessitate the description of novel cases to expand the scope of PTHS.

At present, mutations in a single gene (TCF4) are known to cause PTHS. Our study revealed four cases in which patients showed unique de novo mutations in TCF4 on WES. These patients had a splice site mutation in intron 15, a new frameshift mutation in exon 18, and two new missense mutations in exon 19 (Fig. 3, Table 2). According to the guidelines published by the American College of Medical Genetics and Genomics (ACMG) [11], each variant was categorized as class 4 (PM2, PS2), a likely pathogenic variant. Segregation analysis revealed that the variant had newly spread within the family, which is consistent with the autosomal dominant (AD) inheritance pattern. A diagnosis of a singular genetic disease requires the fulfillment of four criteria, namely, gene, variation, inheritance pattern, and clinical manifestation, all of which were fulfilled for the patients in this study.

Fig. 3 [Images not available. See PDF.]

Schema of the TCF4 gene with localization of novel and previously published mutations in our series

Table 2. A summary of TCF4 gene mutation reported in our series

N°mut	Sex	Exon/Intron	cDNA	Protein	ACMG guidelines	References
1	F	Intron 15	c.1452 + 3A > G		Likely pathogenic	Zweier et al. [10]
2	F	Exon 19	c.2147C > T	p.Ala716Val	Likely pathogenic	Pontual et al. [11]
3	F	Exon 19	c.2026A > G	p.Asn676Asp	Likely pathogenic	This series
4	M	Exon 18	c.1942delA	p.Arg648GlyfsTer12	Likely pathogenic	This series

Although there is currently no specific, universally effective treatment, for individuals with PTHS [2, 23], many studies suggest that early detection and intervention can improve the prognosis of those affected by PTHS [12, 18]. In our study, Patient 4 received rehabilitation training at a her very young age (8 months) and achieved significant improvements, such as the ability to balance while sitting alone and greater cognition, in the following year. Similarly, Patient 2 also received rehabilitation training. As genetic testing becomes increasingly common, a growing number of patients will be given early and accurate diagnoses and promptly advised on the necessary interventions to enhance their treatment outcomes.

TCF4 is a transcription factor that regulates hundreds to thousands of different genes, the mutation of which can cause variable clinical presentations. Most patients with PTHS follow a typical pattern of clinical manifestations, but each individual has slightly different clinical manifestations due to the complex functions of TCF4. Research on the cellular and molecular levels of altered genes is beneficial for identifying phenotypic correlations and examining their functions. TCF4 is disrupted from the beginning of development in PTHS, leading to many developmental abnormalities. In principle, preventing these abnormalities by restoring normal TCF4 expression as early as possible is the best treatment strategy. Research on TCF4 in relation to PTHSs is in relatively early stages, but as more patients are identified and characterized, confidence in genotype/phenotype correlations and understanding of the functions of causal genes may increase.

Conclusions

In conclusion, we describe four PTHS patients with overlapping clinical features but different pathogenic mutations. Our research expands the known list of mutations in PTHS by revealing a new missense mutation and a new frameshift mutation in TCF4 in two Chinese children suffering from PTHS. Patient outcomes can improve with early diagnosis and swift intervention. However, functional analyses to determine how mutated genes affect the function of proteins and to confirm variant causality are lacking. Furthermore, additional studies are warranted to comprehensively understand the varying phenotypes and pathogenesis mechanisms involved. This understanding will enable the development of more effective medication and treatment approaches to manage and combat this rare disease.

Acknowledgements

The research received technical assistance from Kangso Medical Inspection Co., Beijing, China. We wish to extend our appreciation to the patient and her family for their collaboration.

Author contributions

Every writer had a role in conceiving and designing the study. Material preparation, data collection and analysis were performed by Mei Hou, Dianrong Sun and Zongbo Chen. The first draft of the manuscript was written by Dandan Jiang. Danni Jiang and all the other authors commented on previous versions of the manuscript. Every writer reviewed and approved the final manuscript.

Funding

The authors affirm that they did not receive any funding, grants, or other forms of support while preparing this manuscript.

Availability of data and materials

All the data generated or analyzed during this study are included in this published article [and its supplementary information files].

Declarations

Ethics approval and consent to participate

All procedures in the study involving human subjects complied with the ethical guidelines set forth by the Ethics Review Committee of the Affiliated Hospital of Qingdao University. Ethics approval No: QYFY WZLL 28118.

Consent for publication

Before proceeding with the DNA studies and photographing the patient for publication, we obtained signed informed consent from the parents. The writers confirm that the individuals participating in the research gave their informed consent for the publication of their personal details, the visuals in Figs. 1, 2, and 3, and the information in Table 1.

Competing interests

The authors declare that they have no significant financial or nonfinancial conflicts of interest to disclose.

Abbreviations

PTHS

Pitt–Hopkins syndrome

TCF4

Transcription factor 4

CNS

Central nervous system

bGLH

Basic helix-loop-helix

WES

Whole exome sequencing

HGMD

Human Gene Mutation Database

DQ

Developmental quotient

GDS

Gesell developmental scale

ASQ-SE-2

The second edition of the Age and Steps Questionnaire of Social and Emotional Development

ASQ-3

The third edition of the Child Development Screening Test

MRI

Magnetic resonance imaging

EEG

Electroencephalography

AIMS

Alberta Infant Motor Scale

RDs

Rare diseases

ACMG

American College of Medical Genetics and Genomics

AD

Autosomal dominant

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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