BHLHE22 monoallelic and biallelic variants cause a neurodevelopmental disorder with agenesis of the corpus callosum, intellectual disability, abnormal muscle tone, and movement abnormalities

Abstract

Background

BHLHE22 encodes a basic helix-loop-helix transcription factor (bHLH) expressed exclusively in the retina and central nervous system (CNS), and functions as an important regulator of neuronal differentiation. However, BHLHE22 has not yet been associated with a Mendelian neurodevelopmental or neurological disorder.

Methods

Fifteen individuals from thirteen unrelated families carrying BHLHE22 variants identified by exome sequencing were collected through an international collaboration.

Results

De novo missense variants located in the highly conserved helix-loop-helix domain of the protein were found in six individuals, and one recurrent homozygous frameshift variant, NP_689627.1:p.Gly74AlafsTer18, was found in nine individuals. Frequent clinical features include absent or limited speech (10/13), delayed or impaired motor abilities (11/13), intellectual disability (9/12), partial or complete agenesis of the corpus callosum (12/15), involuntary movements and/or stereotypies (11/13), and abnormal muscle tone (13/13), depending on data availability. Two individuals developed spastic paraplegia, without intellectual disability (ID) or callosal anomalies. One individual had moderate developmental delay and ID, but without callosal anomalies.

Conclusion

Collectively, our data establish BHLHE22 as a previously unrecognized neurodevelopmental disease gene. Disruption of BHLHE22, through either dominant or recessive variants, results in a distinct syndrome characterized by abnormalities in brain development, cognition, tone, and movement.

INTRODUCTION

BHLHE22 maps to cytogenetic locus 8q12.3 and encodes a putative 381-amino acid protein with a molecular weight of 36.9 kD from a single exon. The protein contains a N-terminal proline-rich domain, a glycine-rich domain, a polyglycine-serine region, a helix-loop-helix (HLH) domain, and a C-terminal alanine-rich region.¹ BHLHE22 belongs to the basic helix-loop-helix family of transcription factors, characterized by two alpha-helices that mediate dimerization, and an active domain that directs deoxyribonucleic acid (DNA) binding to the E-box.² This superfamily of dimeric transcriptional regulators is an important promoter of cell fate determination, proliferation, and differentiation in CNS development.^1,3,4

Basic helix-loop-helix (bHLH) proteins have been broadly classified based on tissue distribution. Class I bHLH proteins are ubiquitously expressed and bind exclusively to the E-box site. Class II bHLH proteins show tissue-specific expression,² and include BHLHE22, present exclusively in the CNS and retina.^1,5-8 Despite its fundamental role in orchestrating neural development, BHLHE22 variants have not yet been established in association with a human neurodevelopmental disorder.

Corpus callosum dysgenesis (CCD) affects 3.3 in 10,000 births,^9,10 and is linked to a wide range of neurodevelopmental, cognitive, and behavioral issues.¹¹ While hundreds of single genes variants have been associated with early fetal cortical and callosal disruption, many genetic causes remain undiscovered.^12,13 In a trio exome sequencing (ES) cohort of over 600 research participants with CCD, we identified an index case with a rare de novo variant in BHLHE22, NP_689627.1:p.Glu251Gln. Through international collaborations, we identified fourteen additional individuals with overlapping features.

In this study, we report fifteen individuals with either monoallelic de novo or inherited biallelic BHLHE22 variants. The shared clinical and neuroimaging features across these individuals support the identification of BHLHE22 as a novel gene associated with a neurodevelopmental disorder characterized by callosal anomalies and neurological manifestations.

PARTICIPANTS AND METHODS

Participants

Individual 1 was enrolled in the Disorders of Cerebral Development: A Phenotypic and Genetic Analysis Study at the Brain Development Research Program of the University of California, San Francisco (UCSF) as part of a larger cohort study of participants with corpus callosum abnormalities. This study involves human participants and was approved by an UCSF Institutional Review Board (IRB; Study #10-01008). Additional individuals were identified through clinical diagnostic testing and/or data-sharing platforms, including Matchmaker Exchange, ¹⁴ and were evaluated using de-identified data. Individual 6 was enrolled through the Undiagnosed Diseases Network (UDN); UND collaborators are listed in Appendix 1. In accordance with institutional policies, these cases did not require separate IRB approval.

All data were handled in accordance with relevant ethical guidelines and regulations. Written informed consent for participation and publication was obtained from the patients’ legal guardian(s) by the attending geneticist or referring physician. The study was conducted in accordance with the principles of the Declaration of Helsinki.

Available medical records and brain imaging scans/reports were reviewed by the UCSF research team, including a pediatric neuroradiologist and pediatric neurologist. Age ranges were reported instead of exact ages to preserve participant confidentiality. The median age excluding the fetal cases is 11.1 years with a standard deviation of 7.5 years.

Sequencing

Exome or genome sequencing was performed using Illumina platforms (NextSeq 500, Illumina NovaSeq 6000, Illumina NovaSeqX, or Illumina HiSeq 4000). Individuals 7 and 8 were tested through the genetic testing platform GeneDx as previously described.¹⁵ All samples underwent identity quality assurance checks. Bioinformatics analyses were done according to the GATK Best Practices framework. Bidirectional sequences were assembled, aligned to reference gene sequences based on human genome build (GRCh37/hg19 or GRCh38/hg38) and variant calling was performed using GATK HaplotypeCaller (v3.7).

Variant prioritization focused on coding and canonical splice-site variants and incorporated filtering against population databases (gnomAD)¹⁶, retaining rare variants (minor allele frequency [MAF] <0.1% for heterozygous variants and <1% for variants consistent with recessive inheritance) with adequate sequencing coverage (>20 reads). Candidate variants were further assessed based on predicted functional impact, inheritance pattern, and clinical relevance to the phenotype.

For individuals 1, 3, 13, 14, and 15, sequence validation and segregation analysis for the candidate variants were performed through Sanger sequencing. All variants were verified through VariantValidator.¹⁷

RESULTS

We report fifteen individuals (seven females and eight males) in whom clinical, research exome, or genome sequencing was performed to investigate the cause of prenatal anomalies, neurodevelopmental, or neurological disorders, leading to the identification of ultra-rare BHLHE22 variants. Summary and detailed clinical and genetic findings for the cohort are provided (Table 1, Supplementary Table 1).

Table 1. Summary of clinical and genetic features in individuals with BHLHE22 variants.

Data are shown as n/N, where N reflects the number of individuals for whom data were available. ACC, agenesis of the corpus callosum

Feature	Heterozygous variants (n=6)	Homozygous variants (n=9)
BHLHE22 variants	p.Glu251Gln, p.Met255Arg (×2), p.Leu262Pro, p.Ile283Asn, p.Ala289Gly	p.Gly74AlafsTer18 (×9)
Coding impact	Missense	Frameshift
Zygosity	Heterozygous (de novo)	Homozygous
Consanguinity	0/6	7/7
Sex	4 males, 2 females	4 males, 5 females
Age range	0-20 years	0-25 years
Global developmental delay	Moderate-severe (4/6), absent (2/6)	Severe-profound (7/7)
Gross motor ability	Walking ≤3 years (5/6)	Non-ambulatory (7/7)
Language ability	Limited (3/6), normal (3/6)	Non-verbal or vocalizing (7/7)
Intellectual disability	4/6	6/6
Epilepsy	0/6	4/7
Autism traits	4/6	0/5
Stereotypies	4/6	5/7
Hyperkinetic movement disorder	3/6	5/7
Tone abnormalities	Hypotonia (4/6), spasticity (2/6)	Spasticity (7/7)
Brain MRI findings	Partial/complete ACC (3/6)	Complete ACC (9/9)
Postnatal microcephaly	0/4	7/7
Craniofacial dysmorphism	0/6	6/9
Strabismus	3/6	2/7
Gastrointestinal issues	2/6	4/6
Cardiovascular anomalies	0/6	1/9

Heterozygous Variants

Six individuals (1-6) with BHLHE22 de novo missense variants presented with a range of neurodevelopmental and neurological features with or without structural brain anomalies (Figure 1). All were born at term to non-consanguineous parents, with birth weights and lengths within normal limits.

Figure 1. Sagittal T1 images of individuals with BHLHE22-associated disorder showing agenesis of the corpus callosum.

(A) Individual 1 at 16-20 months with partial agenesis of the corpus callosum (ACC) and absence of the rostrum, posterior body, and splenium. (B) Individual 2 at 1-5 months with complete ACC and a small anterior commissure (AC). (C) Individual 8 at 11-15 years with complete ACC and a small AC. (C) Individual 11 at 1-5 years with complete ACC.

Individuals 1 through 3 (p.Glu251Gln in Individual 1, and p.Met255Arg in Individual 2 and 3), exhibited moderate to severe gross and fine motor milestone delays. However, walking was achieved by age 3 in 2/3 individuals. Speech acquisition was also delayed, with limited or absent intentional word use by age 3, and one individual able to speak 2-3 words by age 1.5. All demonstrated autistic characteristics, manifesting as stereotypies, social interaction difficulties, and repetitive self-injurious behaviors. Among two individuals sufficiently old for formal evaluation, both had ID. Brain MRI in infancy revealed isolated partial agenesis of the corpus callosum (ACC) in one individual and isolated complete ACC in the other two (Figure 1). One experienced generalized tonic-clonic seizures in infancy, although no further seizures were reported. All three had central hypotonia, and strabismus, accompanied by additional visual anomalies in one (astigmatism and marked hyperopia). Individual 4 (p.Leu262Pro) had only mild gross motor and language delay, and developed lower limb spastic paraparesis, with symptoms such as claw toes and lower limb pain starting at 13 years old. Despite attending regular education, he experienced difficulties with concentration, dyslexia, and dyspraxia. Individual 5 (p.Ile283Asn) had moderate developmental delay marked by limited language and significantly diminished speech intelligibility. Gross and fine motor abilities were normal, but hypotonia, multiple motor stereotypies, and aggressive behaviors were present. Individual 6 (p.Ala289Gly) had mild motor delay, but normal language and cognitive development. She was diagnosed with spastic paraplegia, with onset at age 3 with initially slowly progression and then stabilized with symptoms of increased reflexes and tone in the leg. She was able to walk with assistance. She had articulation difficulties, suspected to be related to pharyngeal spasticity. Macrocephaly was noted in infancy. A maternally inherited, likely pathogenic variant in BRWD3 was also identified, most consistent with carrier status for ID disorder, X-linked 93 (MIM: 300659); it is possible this variant contributes to her history of macrocephaly, shared by the mother. X-inactivation studies on blood did not show significant skewing.

A schematic representation of the variant locations, tolerance landscape plot, and protein sequence of human BHLHE22 and its vertebrate orthologs is outlined (Figure 2). All six missense variants are absent from gnomAD or other public genomic databases, and located in the HLH domain, crucial for protein dimerization, and highly intolerant to missense variations (Figure 2B). All affected residues are fully evolutionarily conserved from human to zebrafish (Figure 2C), and all variants were characterized by high REVEL scores (0.92-0.95), strongly supporting a deleterious impact. The variants identified in this study have been submitted to the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/). Accession numbers are provided in Supplementary Table 1.

Figure 2. Structural, evolutionary, and intolerance landscape of BHLHE22 highlighting disease-associated variants across conserved and functionally constrained regions.

(A) Tolerance landscape plot of the BHLHE22 protein provided by MetaDome (https://stuart.radboudumc.nl/metadome/). The tool identifies regions of low tolerance to missense variations based on the local ratio of non-synonymous to synonymous variants derived from gnomAD. All de novo variants in our cohort are contained in intolerant/highly intolerant regions (in red) of the landscape.

(B) Schematic representation of the BHLHE22 protein illustrating that all the heterozygous variants are contained in the HLH active domain.

(C) Multiple sequence alignment generated using the University of California, Santa Cruz genome browser (https://genome.ucsc.edu/) showing conservation of the human BHLHE22 protein sequence across vertebrate orthologs. Mutated residues are shown in red.

Homozygous Variants

Nine individuals (7-15) from seven unrelated families with identical biallelic homozygous frameshift variants p.Gly74AlafsTer18, all presented with a severe neurodevelopmental phenotype. Neuroimaging revealed complete or partial ACC in all individuals, often with colpocephaly and absent or hypoplastic anterior and hippocampal commissures (Figure 1). Motor and cognitive impairments were uniformly severe, with 6/7 non-verbal and only one individual able to vocalize. Among individuals of appropriate age for formal assessment, profound intellectual disability was reported in all cases. Early generalized hypotonia was a common feature, evolving into spasticity, dystonia, or a combination of mixed tone abnormalities in all seven living individuals. Most displayed movement disorders, such as choreiform or dyskinetic movements and stereotypies, including hand clenching and lack of purposeful hand use. Epilepsy was present in several individuals, ranging from isolated seizures to Lennox-Gastaut syndrome and EEG abnormalities, including occipital spikes, focal epileptiform activity, and background slowing, were observed even in the absence of clinical seizures. Ophthalmologic abnormalities were reported in 6/13 individuals, most commonly as strabismus/esotropia (4/13). Less frequent findings included refractive errors (2/13), optic atrophy (1/13), retinal detachment with leukocoria (1/13), and cortical visual impairment (1/13). Postnatal microcephaly was observed in all individuals. Craniofacial features included flat facial profile, bifid uvula, cleft palate, microretrognathia, synophrys, low-set ears, epicanthus, depressed nasal root, anteverted nares, bulbous nasal tip, and underfolded helices. Systemic comorbidities were also present. Several individuals had feeding difficulties, dysphagia, or were dependent on gastrostomy tubes. One exhibited cardiac malformations and unilateral renal agenesis. Three individuals developed scoliosis and one had skeletal anomalies including overlapping toes and hypoplastic digits.

All individuals with available information for the recurrent biallelic frameshift variant were born from consanguineous parents and in several cases, chromosomal microarray testing revealed regions of homozygosity. In each case, ES identified a homozygous frameshift variant in BHLHE22 (p.Gly74AlafsTer18), absent from all population databases in the homozygous state, but present in the heterozygous state. In 3/5 families with available information, unaffected parents and siblings were confirmed heterozygous. Two fetal siblings of Individual 10 were both diagnosed prenatally with complete ACC and were found to carry the recurrent homozygous BHLHE22 variant (Individuals 11 and 12); both pregnancies were electively terminated following this diagnosis. The asymptomatic mother was identified as heterozygous for the recurrent frameshift variant in conjunction with a BHLHE22 inframe insertion (p.Ser223_Gly224dup), which has a gnomAD allele frequency of 0.0962 and was not present in any other family member. There was an additional pregnancy, in which prenatal ultrasound showed a normal corpus callosum and postnatally, the infant displayed normal early development. Sequencing confirmed the child was heterozygous for the Gly74AlafsTer18 variant, consistent with recessive inheritance and strongly supporting the pathogenicity of biallelic loss-of-function BHLHE22 variants (Supplementary Figure 1). The family’s pedigree is outlined (Supplementary Figure 2).

Systematic evaluation of ES data did not identify additional pathogenic or likely pathogenic variants to support a second molecular diagnosis in any individual with the recurrent variant. The seven affected families with Punjabi, Caucasian, Syrian, and Turkish ancestries share the same homozygous variant. However, haplotype analysis was not performed and therefore, a founder origin cannot be confirmed.

DISCUSSION

We report 15 individuals from 13 unrelated families with monoallelic missense or biallelic truncating variants in BHLHE22. De novo missense variants were localized in the conserved HLH domain and associated with variable neurodevelopmental outcomes with no evident association between the nature of the amino acid substitution and the observed clinical severity. In contrast, biallelic truncating variants resulted in a consistent severe phenotype characterized by ACC, profound developmental impairment, microcephaly, and mixed tone abnormalities. ES did not identify any additional pathogenic or likely pathogenic variants in any of the 15 individuals to support a second molecular diagnosis explaining spasticity, increased severity, or phenotypic variability. These findings establish BHLHE22 as a neurodevelopmental disease gene with both dominant and recessive inheritance patterns.

Studies in animal models provide biological support for the phenotypes observed in our human cohort. BHLHE22 forms a transcriptional repressor complex with PR/SET Domain 8 (PRDM8) to regulate genes involved in neuronal differentiation and axonal guidance,⁷ including cadherin 11-mediated (CDH11) pathways critical for neural circuit assembly. Variants in PRDM8 and CDH11 have been linked to intellectual disability, seizures, and movement disorders in humans (MIM 616640, MIM 211380 and 619736).

In mice, loss of Bhlhe22 results in abnormal corticospinal neuron projections with premature termination of pyramidal tract axons.¹⁹ This finding parallels the pyramidal signs observed in affected individuals, suggesting that impaired corticospinal tract development represents core pathogenic mechanisms. Additionally, stereotyped scratching behavior reported in Bhlhe22 and Prdm8 null mice may reflect altered motor circuit regulation, consistent with the movement abnormalities seen in our human cohort.⁷

BHLHE22 has also been implicated in the development of amacrine and bipolar neuronal subtypes during retinogenesis.⁶ Consistently, several individuals in our cohort exhibited visual and oculomotor abnormalities, including optic nerve involvement and refractive errors, further supporting a role for BHLHE22 in retinal and visual pathway development.

Significantly, in mouse models, loss of Bhlhe22 in the dorsal telencephalon resulted in almost complete loss of the major interhemispheric commissures in all animals tested: the corpus callosum, the hippocampal commissure, and the anterior commissure. Loss of Bhlhe22 interacting partner Prdm8 produced similar findings, although with mildly reduced penetrance. This closely parallels the neuroradiological findings in our human cohort, in which ACC and other commissural abnormalities were highly penetrant. These concordant human and animal data strongly support a critical role for BHLHE22 in midline commissure formation during early fetal development.

Isolated ACC is often associated with relatively favorable outcomes compared with hypoplastic corpus callosum or callosal anomalies accompanied by additional malformations.¹⁸ In contrast, individuals in our cohort exhibited severe neurodevelopmental impairment, suggesting that disruption of BHLHE22 affects broader neurodevelopmental pathways beyond commissural formation alone. Notably, isolated ACC has one of the lowest diagnostic yields among corpus callosum disorders,¹³ even after trio ES, underscoring the importance of identifying additional genetic causes. Establishing BHLHE22 as a disease gene may therefore improve diagnostic evaluation and prognostic counseling for affected families.

Pathogenic variants in class I helix-loop-helix (bHLH) transcription factors, including TCF4 and TCF12, are well established causes of neurodevelopmental disorders with structural brain abnormalities (MIM:615314, MIM:619718, MIM:610954). Emerging evidence for class II bHLH factors such as BHLHE22 further supports the importance of this transcriptional network in human brain development. Future studies should investigate the functional consequences of HLH-domain missense variants to clarify whether pathogenicity arises through haploinsufficiency, dominant-negative, or gain-of-function mechanisms. Patient-derived cellular models and transcriptomic analyses may further define downstream pathways disrupted by BHLHE22 deficiency.

The same homozygous variant was identified in seven unrelated families with diverse reported ancestries (Punjabi, Caucasian, Syrian, and Turkish). While this raises the possibility of either a shared ancestral allele or recurrent mutational events, haplotype analysis was not performed and therefore, a common origin cannot be established. The variant is very rare in population databases (gnomAD v4) and only observed in the heterozygous state (maximum allele frequency 3.52×10⁻⁵) supporting its compatibility with a recessive disease allele.

In summary, our findings establish BHLHE22 as a neurodevelopmental disease gene associated with both dominant and recessive inheritance patterns and agenesis of brain commissures with a spectrum of motor and cognitive delays. The consistent involvement of the corpus callosum and motor pathways highlights a critical role for BHLHE22 in human midline and corticospinal tract development. Future studies are warranted to define the molecular consequences of pathogenic variants and to further characterize genotype-phenotype correlations, which may ultimately inform prognosis and clinical management.

KEY MESSAGES.

• What is already known on this topic - Corpus callosum dysgenesis (CCD) is among the most common developmental brain disorders. Diagnostic yield even after exome sequencing (ES) is currently below 50%, with many more single genes to be associated with CCD.

• What this study adds - We report on fifteen individuals with BHLHE22 variants linked to a previously unidentified autosomal dominant and recessive neurodevelopmental disorder with corpus callosum agenesis and spasticity.

• How this study might affect research, practice or policy - A molecular diagnosis has the potential to improve early intervention and clinical management. Exome sequencing may be used to clinically identify BHLHE22 variants associated with callosal agenesis, intellectual disability, and spasticity.

Data Availability Statement:

The authors confirm that the data supporting the findings of this study are available within the article and its supplementary data. Consent from individual patients preclude access to the raw genetic data.