Germline de novo alterations of RUNX1T1 in individuals with neurodevelopmental and congenital anomalies

Summary

Runt-related transcription factor 1 translocated to 1 (RUNX1T1; also known as eight-twenty-one [ETO]) encodes a transcription regulator for hematopoietic genes and is well known for its involvement in hematologic malignancies, particularly acute myeloid leukemia (AML). However, its role in congenital disease is less understood. This study provides detailed clinical and molecular information on three cases exhibiting neurodevelopmental and congenital anomalies with germline de novo alterations in RUNX1T1. One case features a de novo nonsense variant in the 5′ region of the gene (c.106C>T p.Gln36Ter), while the other two harbor de novo missense variants in the C terminus end (c.1234G>A p.Gly412Arg and c.1561C>T p.His521Tyr). Common features across cases include craniofacial dysmorphism and neurodevelopmental issues, including developmental delay, learning disabilities, attention-deficit hyperactivity disorder, and autism. This study, in conjunction with previously reported germline disruptions of RUNX1T1, provides evidence supporting the role of germline RUNX1T1 variation in human congenital neurodevelopmental disorders.

While RUNX1T1 is well known for its role in leukemia, its involvement in congenital disease is less understood. We present detailed clinical and molecular information on three individuals exhibiting neurodevelopmental and congenital anomalies with germline de novo alterations in RUNX1T1. Further research is needed to clarify a potential association.

Introduction

RUNX1T1 (Runt-related transcription factor 1 translocated to 1; MIM: 133435), also known as ETO (eight-twenty-one), is a member of the core-binding transcription factor complex, which plays a critical role in the regulation of hematopoietic gene transcription. It is best known for its role in hematologic malignancies, particularly acute myeloid leukemia (AML). In AML, the t(8;21) chromosomal translocation results in the fusion of RUNX1T1 with RUNX1 (AML1), leading to aberrant transcriptional regulation and disruption of normal hematopoiesis. This fusion characterizes a specific subtype of AML, known as AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1.¹

Despite its well-established role in leukemia, the involvement of RUNX1T1 in constitutional disease is less understood. Emerging evidence suggests that genetic alterations in RUNX1T1 may result in a range of neurodevelopmental and congenital anomalies. This insight is supported by earlier reports of disruption of RUNX1T1 in subjects with congenital heart disease and intellectual disabilities,^2,3 as well as more recent identification of de novo variants in this gene within large cohorts of individuals with neurodevelopmental disorders.^4,5,6,7 While experimental studies to support a possible disease association are limited, RUNX1T1 is known to be highly expressed in brain and heart,^3,8 suggesting that the gene warrants further investigation as a candidate gene for neurodevelopmental disorders.

Here, we present detailed clinical and genetic data from three individuals identified with de novo variants in RUNX1T1. These cases illustrate a diverse array of phenotypic manifestations, ranging from developmental delay and speech impairments to distinct craniofacial and physical anomalies. By delineating the clinical features associated with RUNX1T1 variants, this report aims to enhance the understanding of the gene’s role in neurodevelopment and its potential impact on clinical practice.

Subjects and methods

Subjects

The cases in this study include three probands who were referred to GeneDx for exome sequencing (ES). All patients were evaluated by a clinical genetics team. Detailed medical histories, physical examinations, and relevant laboratory tests were provided by the referring sites.

Ethics approval and consent to participate

This study was conducted under GeneDx’s research protocol “Research to Expand the Understanding of Genetic Variants: Clinical and Genetic Correlations,” approved by the Western Institutional Review Board (IRB) (protocol 20171030). All research subjects provided written consent to participate. For subject 2, informed written consent was obtained for the use of a photograph.

Exome sequencing

Using genomic DNA from the proband, parents (all subjects), and sibling (subject 2), the exonic regions and flanking splice junctions of the genome were captured using the IDT xGen Exome Research Panel v.1.0 (Integrated DNA Technologies, Coralville, IA) or the Twist Bioscience Exome 2.0 (Twist Biosciences, South San Francisco, CA). Massively parallel (NextGen) sequencing was performed on an Illumina NovaSeq6000 or NovaSeq X Plus with 2 × 150 bp paired-end reads. Reads were aligned to human genome build GRCh37/UCSC hg19 and analyzed for sequence variants using a custom-developed analysis tool. Reported variants were confirmed, if necessary, by Sanger sequencing in the proband and submitted relatives. Additional sequencing technology and variant interpretation protocol has been previously described.⁹ The 2021 assertion criteria for variant classification are publicly available on the GeneDx ClinVar submission page (see web resources).

Results

The study includes three subjects with neurodevelopmental abnormalities and de novo variants in RUNX1T1, where no alternative diagnostic finding was identified via ES or other genetic testing. Table 1 summarizes the clinical features mapped to human phenotype ontology (HPO) terms. Table 2 provides information on the variants identified in each proband. A detailed description of each subject is provided as follows.

Table 1.

Clinical features of the three probands mapped to HPO terms

HPO ID	HPO term	Subject 1 (4 years)	Subject 2 (6 years)	Subject 3 (5 years)
Neuro/developmental findings
HP:0000750	delayed speech and language development	+	+	+
HP:0007018	attention-deficit hyperactivity disorder	–	+	+
HP:0012434	delayed early-childhood social milestone development	+	+	–
HP:0000717	autism	NR	+	+
HP:0000718	aggressive behavior	–	+	NR
HP:0001263	global developmental delay	–	+	+
HP:0010862	delayed fine motor development	+	+	+
HP:0002311	incoordination	–	+	NR
HP:0001250	seizure	–	–	+
HP:0100702	arachnoid cyst	NR	NR	+
HP:0001328	specific learning disability	+	–	+
HP:0001337	tremor	–	+	–
Dysmorphic facial features
HP:0000369	low-set ears	+	+	+
HP:0000494	down-slanted palpebral fissures	+	–	–
HP:0000319	smooth philtrum	+	–	–
HP:0000343	long philtrum	+	–	–
HP:0000219	thin upper lip vermilion	+	–	–
HP:0000475	broad neck	+	–	–
HP:0000470	short neck	+	–	–
HP:0000276	long face	–	+	–
HP:0000316	hypertelorism	+	+	–
HP:0000337	broad forehead	+	–	+
HP:0011246	underdeveloped superior crus of antihelix	+	–	+
HP:0000396	overfolded helix	–	–	+
HP:0000455	broad nasal tip	+	–	+
Other congenital anomalies
HP:0008751	laryngeal cleft	+	NR	NR
HP:0001629	ventricular septal defect	+	NR	NR
HP:0001631	atrial septal defect	+	NR	NR
HP:0005301	persistent left superior vena cava	+	NR	NR
HP:0010296	ankyloglossia	+	–	–
HP:0010609	skin tags	–	+	–
HP:0001763	pes planus	+	+	–
HP:0010047	short fifth metacarpal	–	–	+
Growth
HP:0004325	decreased body weight	–	+	–
HP:0000256	macrocephaly	–	–	+
Medical issues
HP:0002020	gastroesophageal reflux	+	–	–
HP:0001903	anemia	+	–	–
HP:0002360	sleep abnormality	+	+	–
HP:0006482	abnormal dental morphology	NR	+	–

The ages listed in the top row reflect the ages at which the clinical information was obtained. NR, not reported.

Table 2.

De novo sequence variants identified in RUNX1T1 in the probands

Subject	hg19 coordinates	Exon	cDNA change	Protein change	Effect	ClinVar ID	Provean	MutTaster	SIFT	CADD	PhyloP	REVEL
1	chr8:93,029,493	6	c.106C>T	p.Gln36Ter	nonsense	SCV005184299	N/A	N/A	N/A	N/A	N/A	N/A
2	chr8:92,988,166	13	c.1234G>A	p.Gly412Arga	missense	SCV005184298	−4.04	disease causing	0.999	33	0.935	0.862
3	chr8:92,972,643	15	c.1561C>T	p.His521Tyr	missense	SCV005184300	−4.94	disease causing	1	26.7	1.048	0.863

Transcript: NM_001198625.1. The reading frame is on the minus strand. All variants are absent from gnomAD v.2.1.1. All variants are de novo (with confirmed paternity and maternity). Both missense variants are in the C-terminal end of the protein and fully conserved across 100 vertebrate species (UCSC genome browser). As assessed under a research protocol, the in silico predictions for both missense changes are unanimously deleterious according to PROVEAN, MutationTaster, SIFT, CADD, PhyoP, and REVEL. N/A, not applicable.

^aGly412Arg is detected mosaic.

Subject 1

The proband is a 4-year-old male with developmental delay (notably in speech), ventricular septal defect (VSD), atrial septal defect (ASD), persistent left superior vena cava (SVC), and gastroesophageal reflux disease (GERD). He was born at 41+2 weeks gestation following an uncomplicated pregnancy. Infancy was marked by choking concerns, leading to a posterior frenulotomy and laryngeal cleft repair at 7 months, which improved coughing after feeding. Despite walking at 12 months, he had no words at 21 months, with persistent impairments in speech and social skills. Physical examination revealed down-slanting palpebral fissures, a long smooth philtrum, thin upper vermillion, and a broad, short neck. His most recent clinical history includes anemia, which is being treated with iron supplements. Trio ES at 21 months identified a de novo heterozygous nonsense variant in exon 6 of RUNX1T1: c.106C>T (p.Gln36Ter).

At his last assessment, at 4 years of age, the patient had mostly one-word utterances with occasional two-word phrases. His most recent weight, height, and head circumference percentiles are measured to be 42.7%, 20.57%, and 2.5%, respectively.

Subject 2

The proband is a 7-year-old female who presented at 4 years of age with speech and motor developmental delays, a diagnosis of autism spectrum disorder (level 2), attention-deficit hyperactivity disorder (ADHD), and aggressive behaviors. She was born at 37 weeks and 6 days gestation via cesarean section due to breech positioning and a knot in her umbilical cord. Her birth was complicated by gestational hypertension and maternal cytomegalovirus (CMV) positivity, although CMV testing at birth was normal. At 6 months, she experienced tremor episodes, initially concerning for infantile spasms, though an electroencephalogram (EEG) was normal, and her symptoms were assumed to potentially reflect sleep myoclonus. By age 6 years, she had significant sleep difficulties. Her medical history also included a large skin tag on her left ear (removed at birth) and multiple dental crowns and fillings despite good dental hygiene. An evaluation at age 6.5 years highlighted craniofacial features including a long face, low-set ears, and hypertelorism, as well as pes planus (Figure 1). Her most recent weight, height, and head circumference percentiles are measured to be 1.7%, 19%, and 10.71%, respectively.

Figure 1.

Photograph of proband 2

Note the long face, low-set ears, and hypertelorism.

The proband’s family history is significant for one sibling with neurodevelopmental delay and nondiagnostic ES and a second sibling with concerns for short stature with no additional testing.

Prior genomic microarray and fragile X testing in the proband were reported as normal. Trio ES revealed a de novo missense variant in exon 13 of RUNX1T1, c.1234G>A (p.Gly412Arg), with an allele fraction of 31%. Orthogonal Sanger sequencing confirmed the variant to be mosaic. The proband’s sibling with neurodevelopmental delay did not carry this variant.

Subject 3

A 5-year-old female with a history of developmental delay, intellectual disability, macrocephaly, and seizures was evaluated for a range of symptoms. The proband had macrocephaly since birth with a head circumference above the 97^th percentile and delayed development noted at 4 months of age due to poor head control. Although other motor milestones were met, she had language delays at 2 years of age, articulating fewer than 20 words with no phrases. A possible seizure was suspected at the same age that re-occurred in later years both with and without fever, and she was diagnosed with epilepsy. At 5 years and 4 months, a comprehensive neuropsychiatric evaluation showed that her intellectual and cognitive skills were in the low average range, with below-average processing speed. She was diagnosed with a general academic learning disorder, speech delay, poor fine motor dexterity, verbal memory deficits, and ADHD.

The latest evaluations at 5 years of age showed the head circumference to be above the 97^th percentile for her age, a broad forehead, a broad nasal tip, short fifth digits bilaterally, and low-set ears with underdevelopment of the superior crus on the right and an overfolded helix on the left. The muscle tone and bulk were normal. An EEG at this age revealed bursts of spike and slow-wave complexes. Brain imaging using magnetic resonance imaging (MRI) showed a small arachnoid cyst in the cranial fossa, but it was otherwise normal.

Notably, while both parents were noted to have a larger head circumference, there was no history for other features or a genetic condition found in the extended family of the proband.

An initial evaluation using chromosomal microarray, PTEN sequencing, and an autism/intellectual disability panel did not identify an etiology for her features. Trio ES identified a de novo heterozygous missense variant in exon 15 of RUNX1T1: c.1561C>T (p.His521Tyr).

Discussion

The role of RUNX1T1 in congenital disease is not well understood. Reports of the disruption of the gene are limited in the literature, and variants that could potentially cause a loss of function are rare in the general population. In the gnomAD database (v.2.1.1), RUNX1T1 is moderately constrained for both loss-of-function variants and missense changes (pLI = 0.98, misZ = 2.17), suggesting that variation in this gene is not well tolerated in healthy adults and that RUNX1T1 could potentially play an important role in normal human development.

The first documented case of a constitutional disruption of RUNX1T1 involved an individual with intellectual disability, mild craniofacial dysmorphism, and a VSD who had a balanced reciprocal translocation with the karyotype 46,XY,t(5;8)(q33;q22).³ Mapping of the translocation breakpoints using fluorescence in situ hybridization (FISH) probes revealed that the breakpoint on chromosome 8 occurred at intron 1b of RUNX1T1, while the breakpoint on chromosome 5 mapped to a gene-empty region. This disruption was believed to affect the expression of RUNX1T1, potentially explaining the observed phenotype. Subsequently, a de novo deletion of exons 3–7 of RUNX1T1 was reported in a 38-year-old female with mild intellectual and learning disabilities and mild facial anomalies.² Additionally, a full deletion of RUNX1T1 is identified in a proband with intellectual disability, anemia, ASD, and seizures; however, this deletion extends to two adjacent genes, SLC26A7 and TRIQK.¹⁰ The literature features reports of additional de novo sequence variants in RUNX1T1 in cases with intellectual disabilities and autism, though these findings were observed in large cohort studies without detailed phenotypic descriptions. Additionally, some of those cases also have other de novo variants.^4,5,6,7

The three cases presented in this study, along with the three reported in literature,^2,3,10 are currently the only documented instances, to our knowledge, of isolated de novo alterations in RUNX1T1 with detailed phenotyping. Collectively, they provide an overview of the phenotypic spectrum that could potentially be linked to this gene. Although the observed phenotypes are diverse, common features include craniofacial dysmorphism and varying degrees of neurodevelopmental impairments, such as developmental delay, autism, and behavioral anomalies. An additional recurrent feature includes congenital heart defects, which is observed in subject 1 of this study, as well as in the previously published subject with a balanced translocation,³ and the published subject with a three-gene deletion involving RUNX1T1.¹⁰ Neither of the subjects presented here nor those documented in the literature have shown growth abnormalities. Additionally, no instances of microcephaly have been reported; in contrast, one of our subjects presented with macrocephaly. Identifying additional phenotypic features and providing a more comprehensive description of a recognizable phenotype will require the discovery of more subjects with potentially deleterious variants in RUNX1T1. In future studies, standardized physical assessments, utilization of AI-powered dysmorphology technology, and additional diagnostic workups such as genome sequencing could further support the data presented in this and previous reports.

Considering the types of variants, the gene constraint scores, and previous reports of deletions and gene disruptions, it is tempting to propose a loss-of-function mechanism for this gene in disease development. However, it cannot be ruled out that variants with other mechanisms, such as a gain-of-function or dominant-negative effect, may also contribute to the phenotype or result in a phenotype different from those caused by loss of function. Notably, two of the cases presented here carry missense mutations, both of which are in conserved residues in the C terminus end of the protein. Understanding the pathogenesis and disease-causing mechanisms for these variants will require functional analysis and experimental studies.

There are many examples of genes initially identified for their involvement in cancer through somatic alterations, later found to cause congenital anomalies through germline defects. For example, KMT2A, like RUNX1T1, was originally known only for its role in hematological malignancies but was later discovered to contribute to neurodevelopmental disorders.¹¹ These cases highlight the overlap between oncogenic and developmental pathways, emphasizing the importance of studying genes across different biological contexts. This brief communication highlights a potential association between de novo variants in the RUNX1T1 gene and a spectrum of neurodevelopmental and physical anomalies. The findings emphasize the importance of including RUNX1T1 variants in the genetic evaluation of patients with complex neurodevelopmental disorders. Further research is needed to confirm these observations and explore the underlying mechanisms, which could ultimately enhance the diagnosis and management of affected individuals.

Data code and availability

Exome sequence data were generated during clinical testing; however, patients are not consented for data sharing.

Acknowledgments

We would like to thank the families for participating in this study.

Declaration of interests

E.A.-E., D.A.C., E.A.N., Y.Q., K.M., and E.T. are employees of and may own stock in GeneDx, LLC.

Web resources

• CADD, https://cadd.bihealth.org/score

• GeneDx ClinVar submission page, http://www.ncbi.nlm.nih.gov/clinvar/submitters/26957/

• gnomAD v.2.1.1, https://gnomad.broadinstitute.org/

• Human Phenotype Ontology (HPO), https://hpo.jax.org/

• MutationTaster, https://www.mutationtaster.org/

• OMIM, http://www.omim.org/

• PROVEAN, https://www.jcvi.org/research/provean

• SIFT, https://sift.bii.a-star.edu.sg/

• UCSC Genome Browser, http://genome.ucsc.edu/