Bi-allelic variants in neuronal adhesion molecule astrotactin 1 gene ASTN1 cause diverse neurodevelopmental disorders

Jesse M Levine; Daniel G Calame; Riccardo Sangermano; Haowei Du; Ahmed Saad; Jasmin Lisfeld; Tatjana Bierhals; Jonas Denecke; Eyyup Uctepe; Merve Yoldas Celik; Ahmet Yesilyurt; Hilal Yildiz Er; Elif Yilmaz Gulec; Aziza Mushiba; Naif Almontashiri; Pawel Gawlinski; Wojciech Wiszniewski; Ender Karaca; Lama Alabdi; Davut Pehlivan; Dana Marafi; Maha S Zaki; Fowzan S Alkuraya; Joseph G Gleeson; Shalini N Jhangiani; Richard A Gibbs; Jennifer E Posey; Kinga M Bujakowska; James R Lupski

doi:10.1016/j.ajhg.2025.12.011

. 2026 Jan 15;113(2):362–379. doi: 10.1016/j.ajhg.2025.12.011

Bi-allelic variants in neuronal adhesion molecule astrotactin 1 gene ASTN1 cause diverse neurodevelopmental disorders

Jesse M Levine ^1,^2,^28,^∗, Daniel G Calame ^1,^2,²⁸, Riccardo Sangermano ³, Haowei Du ⁴, Ahmed Saad ^4,⁵, Jasmin Lisfeld ⁶, Tatjana Bierhals ⁶, Jonas Denecke ⁷, Eyyup Uctepe ⁸, Merve Yoldas Celik ⁹, Ahmet Yesilyurt ^8,¹⁰, Hilal Yildiz Er ⁸, Elif Yilmaz Gulec ^11,¹², Aziza Mushiba ¹³, Naif Almontashiri ^14,¹⁵, Pawel Gawlinski ¹⁶, Wojciech Wiszniewski ^16,¹⁷, Ender Karaca ^18,¹⁹, Lama Alabdi ²⁰, Davut Pehlivan ^1,², Dana Marafi ^4,²¹, Maha S Zaki ^22,²³, Fowzan S Alkuraya ^20,²⁴, Joseph G Gleeson ^25,²⁶, Shalini N Jhangiani ²⁷, Richard A Gibbs ^4,²⁷, Jennifer E Posey ⁴, Kinga M Bujakowska ³, James R Lupski ^2,^4,^27,^∗∗

¹Section of Pediatric Neurology and Developmental Neuroscience, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

²Texas Children’s Hospital, Houston, TX, USA

³Ocular Genomics Institute, Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, MA, USA

⁴Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA

⁵Medical Molecular Genetics Department, Human Genetics and Genome Research Institute, National Research Centre, Cairo, Egypt

⁶Institute of Human Genetics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

⁷Department of Pediatrics, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

⁸Acibadem Labgen Genetic Diagnosis Center, Istanbul, Türkiye

⁹Department of Pediatric Metabolism, Adana City Training and Research Hospital, Adana, Türkiye

¹⁰Acibadem Maslak Hospital, Istanbul, Türkiye

¹¹Department of Medical Genetics, İstanbul Medeniyet University Medical School, Istanbul, Türkiye

¹²Medical Genetics Clinic, Istanbul Goztepe Prof. Dr. Suleyman Yalcin City Hospital, Istanbul, Türkiye

¹³Section of Medical Genetics, Department of Pediatrics, Children’s Specialist Hospital, King Fahad Medical City, Riyadh, Saudi Arabia

¹⁴College of Applied Medical Sciences and Center for Genetics and Inherited Diseases (CGID), Taibah University, Madinah, Saudi Arabia

¹⁵Research Department, King Khaled Eye Specialist Hospital, Riyadh, Saudi Arabia

¹⁶Department of Medical Genetics, Institute of Mother and Child, Warsaw, Poland

¹⁷Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA

¹⁸Department of Pathology, Baylor University Medical Center, Dallas, TX, USA

¹⁹Texas A&M School of Medicine, Dallas, TX, USA

²⁰Department of Translational Genomics, Center for Genomic Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia

²¹Department of Pediatrics, College of Medicine, Kuwait University, Safat, Kuwait

²²Clinical Genetics Department, Human Genetics and Genome Research Institute, National Research Centre, Cairo, Egypt

²³Medical Genetics Department, Armed Forces College of Medicine (AFCM), Cairo, Egypt

²⁴Lifera Omics, Riyadh, Saudi Arabia

²⁵Department of Neurosciences, University of California, San Diego, La Jolla, CA, USA

²⁶Rady Children’s Institute for Genomic Medicine, San Diego, CA, USA

²⁷Human Genome Sequencing Center, Baylor College of Medicine, Houston, TX, USA

^∗

Corresponding author jesse.levine@bcm.edu

^∗∗

Corresponding author jlupski@bcm.edu

²⁸

These authors contributed equally

PMCID: PMC13087404 NIHMSID: NIHMS2163351 PMID: 41544630

Summary

ASTN1 encodes astrotactin 1, a neuronal-glial ligand in the developing brain that promotes neuronal migration along radial glia in brain structures with laminar organization, such as the cerebral cortex, hippocampus, and cerebellum. In mouse models, disruption of Astn1 results in neuronal migration deficits, a mild reduction in cerebellar volume, and balance and coordination deficits. In humans, bi-allelic ASTN1 variants have been identified in nine individuals with neurodevelopmental disorders (NDDs) with or without brain malformations. ASTN1 additionally interacts with astrotactin 2 (ASTN2) to implement neuronal migration; ASTN2 deletions associate with NDDs with reduced penetrance. Here, we describe eighteen individuals with NDDs from twelve unrelated families with bi-allelic, ultra-rare, predicted damaging variants in ASTN1 and one individual with heterozygous variants in both ASTN1 and ASTN2. We expand the clinical phenotypic descriptions of ASTN1-related NDDs, which range from mild to profound developmental delay or intellectual disability and can be associated with autism, attention-deficient hyperactivity disorder (ADHD), and epilepsy. Other recurrent abnormalities include dysmorphic facial features, hypotonia, spasticity, and ataxia. Additionally, we add to the neuroradiographic phenotype of this condition, which can be normal, mildly dysmorphic (a thin corpus callosum and cerebellar dysgenesis), or severely dysmorphic (polymicrogyria and lissencephaly). Remarkably, three genetic models of multilocus pathogenic variation (MPV), including tri-allelic, double heterozygous, and double homozygous due to distributive absence of heterozygosity (AOH), were observed. This ASTN1 allelic series characterizes the consequences of perturbations in radial-glia-guided neuronal migration in humans, the phenotypic spectrum of ASTN1-related NDDs, and the contribution of MPV to the genetic basis of NDDs.

Keywords: ASTN1, ASTN2, neurodevelopmental disorders, multilocus pathogenic variation, distributive AOH, cerebellum, autism, epilepsy, intellectual disability, neuronal migration

This manuscript firmly establishes ASTN1 as a recessive neurodevelopmental disease gene, broadens the phenotypic spectrum of ASTN1-related neurodevelopmental disorders, provides further evidence for the contribution of splicing aberration to neurodevelopmental disorders, and highlights the contribution of multilocus pathogenic variation to the prevalence of neurodevelopmental disorders.

Introduction

Neural structures with laminar organization, such as the neocortex, cerebellum, and hippocampus, are generated in a predominantly inside-out pattern,¹ with neurons derived from progenitor cells in the ventricular and subventricular zones migrating along radial glial cells.² Neuronal migration in laminar structures is regulated by the gene paralogs ASTN1 (MIM: 600904) and ASTN2 (MIM: 612856), the genes encoding astrotactin 1 (ASTN1) and astrotactin 2 (ASTN2), respectively. ASTN1 is a neuronal-glial ligand expressed on postnatal neurons that interacts with cadherin 2 (CDH2) on neurons and glial cells at the migrational junction to facilitate radial migration of neurons along radial glial cells to their final laminar positions.³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰ ASTN2 regulates the trafficking of ASTN1 to the neuronal surface throughout migration to mediate this ASTN1-dependent adhesion.¹¹ Disruption of neuronal-glial adhesion by blocking ASTN1 impairs neuronal migration, and Astn1 knockout mice demonstrate slowed neuronal migration and abnormal cerebellar Purkinje cell development, ultimately resulting in impaired balance and coordination.¹² While the cerebellum is a key regulator of motor control and coordination, its role in non-motor behaviors and autism is increasingly appreciated.¹³ Likewise, Astn2-knockout mice have altered cerebellar circuit properties¹⁴ and autism-like behaviors.¹⁵

In humans, there is significant constraint against ASTN1 and ASTN2 loss of function (LoF) (ASTN1: pLI [probability of loss-of-function intolerance] = 0.9, LOEUF [loss-of-function observed/expected upper-bound fraction] = 0.47, and pHaplo [probability of haploinsufficiency] = 0.91; ASTN2: pLI = 0.99, LOEUF = 0.47, and pHaplo = 0.88).¹⁶^,¹⁷ Heterozygous ASTN2 deletions have been identified in autism, schizophrenia, and intellectual disability cohorts and are thought to contribute to neurodevelopmental disorder (NDD) risk with reduced penetrance.¹⁸^,¹⁹^,²⁰^,²¹^,²²^,²³^,²⁴^,²⁵^,²⁶^,²⁷ Bi-allelic ASTN1 variants have been reported in nine individuals with NDDs with and without brain malformations,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³ albeit with minimal clinical information. Consequently, the gene-disease association between ASTN1 and NDDs has not been established, as evidenced by the lack of entries in clinical genomics databases such as OMIM, ClinGen, and GenCC.

We report here an ASTN1 allelic series comprising eighteen individuals with NDDs from twelve unrelated families. Seventeen individuals were found to have bi-allelic, ultra-rare, predicted damaging variants in ASTN1, and one individual had heterozygous variants in both ASTN1 and ASTN2. We demonstrate a neurodevelopmental phenotypic spectrum ranging from mild to profound developmental delay/intellectual disability in addition to variable findings including autism, attention-deficient hyperactivity disorder (ADHD), epilepsy, dysmorphic facial features, hypotonia, spasticity, and ataxia. We provide functional evidence for the in situ predicted pathogenicity of three ASTN1 single-nucleotide variants (SNVs) prioritized through SpliceAI via mini-gene splicing assays. Additionally, we characterize three models of multilocus pathogenic variation (MPV) within this study, including tri-allelic, double heterozygous, and double homozygous due to distributive absence of heterozygosity (AOH). Taken together, we establish bi-allelic ASTN1 variants as a cause of autosomal-recessive NDDs, broaden genetic models underlying NDD, contribute an expanded clinical and neuroradiographic phenotypic description of ASTN1-related NDDs, and provide neurobiological insight into the disease process.

Subjects, material, and methods

Participant ascertainment and identification

We initially identified individuals with predominantly bi-allelic ASTN1 (GenBank: NM_004319.3) variants through family-based genomics within the Baylor College of Medicine Genomics Research to Elucidate the Genetics of Rare Diseases (BCM-GREGoR) research NDD cohort. Additional individuals were identified through GeneMatcher and international collaborations. This cohort includes five families (families 7–11) who were previously published in NDD cohort publications²⁸^,²⁹^,³⁰^,³¹; we collected additional detailed clinical phenotypic information on previously published families and added new families to the ASTN1 variant cohort (families 1–6). The study was approved by Baylor College of Medicine institutional review board (IRB) protocol H-29697. Informed consent, including consent to have the results of this research work published, was obtained from all participants as required by the IRB or through the respective IRBs of our collaborators. Phenotypic information, brain magnetic resonance imaging (MRI), and clinical photographs were collected from clinical collaborators when possible.

Genomic sequencing and variant prioritization

Research exome sequencing (ES) for families 3, 7, 8, and 9 was performed as part of the BCM-GREGoR at the Baylor College of Medicine Human Genome Sequencing Center as previously described.²⁸^,³⁰^,³¹^,³⁴ Family 1 underwent clinical ES through Blueprint Genetics. Family 2 underwent research ES as part of a long-standing neurogenetics research program at UCSD.³⁴ Family 4 underwent ES as previously described.³⁵ Family 5 underwent ES at Acibadem Labgen.³⁶ Family 6 underwent ES at the Institute of Human Genetics, Helmholtz Center Munich.³⁷ Families 10 and 11 underwent research ES at the King Faisal Specialist Hospital and Research Center.²⁹^,³² Variant prioritization was performed as previously described using in silico predictions of pathogenicity, gnomAD v.2.1.1 allele frequencies, and internal database allele frequencies.³⁴ Additionally, rare potential pathogenic splicing variants were screened using SpliceAI within the BCM-GREGoR database.³⁸ The filter criteria included variants with fewer than 20 alleles in approximately 12,000 internal ES data samples, a frequency of less than 0.001 in gnomAD v.2.1.1, and a SpliceAI Δscore above 0.8 for high-confidence splice variants. Variants were orthogonally confirmed by Sanger dideoxy sequencing whenever possible.

Functional mini-gene splicing assay

A mini-gene splicing assay was performed using a mini-gene split GFP construct,³⁹ in which the N- and C-terminal parts of the GFP gene were separated by SMN1 introns 7 and 8 (GenBank: NM_000344). Reference and mutated gene fragments containing 500–1,000 bp, including and surrounding the studied exons of the ASTN1 gene flanked with 32 bp vector homology arms, were synthetized (TWIST Bioscience, USA) and cloned into the mini-gene construct (Gibson Assembly Master Mix, New England Biolabs). After Sanger sequencing verification of all constructs, they were transfected into the HEK293 cells (Lipofectamine 3000, Thermo Fisher Scientific). Forty-eight hours post-transfection, total RNA was extracted from the transfected cells (RNAeasy Mini Kit, Qiagen), and cDNA was generated using random hexamer primers (SuperScript IV Synthesis Kit, Thermo Fisher Scientific). Subsequently, the mini-gene transcripts were amplified from the cDNA using primers specific to the split GFP fragments (F: 5′-CACACTGGTGACAACATTTACATAC-3′; R: 5′-GAAATCGTGCTGTTTCATGTGATC-3′). The PCR products were visualized by gel electrophoresis, and the PCR products were either isolated from the gel (Gel DNA Recovery Kit, Zymo Research) or column purified from the PCR mix (DNA Clean & Concentrator-5, Zymo Research). The PCR products were analyzed by Sanger sequencing or by next-generation amplicon sequencing (MiSeq, Illumina, OGI Genomics Core). The splicing pattern analysis was performed by aligning the sequence reads to the hybrid reference of the split GFP construct containing the SMN1 introns and the studied exons and flanking introns of ASTN1 (STAR Aligner)⁴⁰ and visualizing the reads in the Integrated Genome Viewer (IGV).

AOH calculations

Unphased SNVs from ES data were used to call AOH genomic intervals, a surrogate measurement of runs of homozygosity (ROHs), using BafCalculator, an in-house tool developed by Eldomery et al. (available at https://github.com/BCM-Lupskilab/BafCalculator).⁴¹ Briefly, B allele frequencies (BAFs) were calculated for each SNV, and the absolute value of each BAF minus 0.5 was calculated. Genomic segments were classified as AOH regions if the mean-transformed BAF value within the region was greater than 0.47 and if the segmental size exceeded 50 kb. The total AOH size was calculated by adding up the total sum of AOH identified in the autosomes.

Results

Identification and phenotypic characterization of individuals with NDDs likely due to ASTN1 variants

Here, we add six unreported families (families 1–6) to the five previously reported families (families 7–11) containing individuals with variants in ASTN1. Additionally, we incorporate new extended family members (individuals 14 and 15) from one previously reported family (family 11) (Figure 1; Table 1). We have also included a 12^th family, which was recently reported.³³ Ten of the twelve families include consanguinity by report (families 1, 2, 3, 4, 5, 7, 9, 10, 11, and 12).

Eighteen individuals from twelve unrelated families with variants in *ASTN1* demonstrating three genetic models for multilocus pathogenic variation

Pedigrees of twelve families of individuals with predicted damaging variants in *ASTN1.* Families 1, 2, 3, 4, 5, 9, 10, and 11 and the 12^th family from a recent case report display homozygous variants in *ASTN1*, while the individuals in families 6 and 8 have compound heterozygous variants in *ASTN1.* Three families in this cohort demonstrate three models of multilocus pathogenic variation (MPV). Family 1 (teal box) has tri-allelic MPV, with homozygous variants in *ASTN1* and a heterozygous *de novo* variant in *KDM3A.* Family 3 (orange box) shows double homozygous variants due to distributive AOH, with homozygous variants in both *ASTN1* and *TRAK2.* The pattern of AOH, marked by gray zones, for the affected individual in family 3 is shown from chromosomes 1 and 2, with *ASTN1* and *TRAK2* falling within regions of AOH (red lines). Family 7 (purple box) is an example of double-heterozygous MPV, with heterozygous variants in two genes known to interact to contribute to typical neurodevelopment, *ASTN1* and *ASTN2.* The country of origin of each family is in green text. Citations for previously reported families are in purple text.

Table 1.

Summary of ASTN1 variant alleles

Family	F1	F2		F3	F4	F5	F6		F7	F8		F9		F10		F11				Ayaz et al.³³
Individual	P1	P2	P3	P4	P5	P6	P7		P8	P9		P10	P11	P12	P13	P14	P15	P16	P17	N/A
Nucleotide	c.1523+1G>T	c.1132C>T		c.1736+5G>A	c.1599−14G>A	c.1270+1G>A	c.838_839delGA	c.3125delAinsGA CCACAAGTG	c.2773A>G (htz)	c.3283A>C (htz)	c.2770C>T (htz)	c.2224G>C		c.3159_3160del		c.3334C>T				c.1549C>T
Protein	p.?	p.Arg378Ter		p.?	p.?	p.?	p.Glu280LysfsTer11	p.Glu1042GlyfsTer28	p.Met925Val	p.Met1095Leu	p.His924Tyr	p.Gly742Arg		p.Gln1053HisfsTer13		p.Arg1112Ter				p.Arg517Ter
Genetic model	homozygous (tri-allelic MPV)	homozygous		double homozygous due to distributive AOH MPV	homozygous	homozygous	compound heterozygous	compound heterozygous	double heterozygous MPV	compound heterozygous	compound heterozygous	homozygous		homozygous		homozygous				homozygous
CADD	34	37		18.45	9.272	33	N/A	N/A	12.77	23.2	23.2	23.7		N/A		38				42
REVEL	N/A	N/A		N/A	N/A	N/A	N/A	N/A	0.091	0.244	0.136	0.254		N/A		N/A				N/A
SpliceAI	DL Δ 0.95	N/A		DL Δ 0.81	AL Δ 0.5, AG Δ 0.35	DL Δ 0.99, DG Δ 0.63	N/A	N/A	N/A	N/A	N/A	N/A		N/A		N/A				N/A
phyloP100way	7.155	2.334		6.173	0.428	7.536	7.516	7.52	1.38	7.603	7.314	5.614		5.812		3.184				3.391
gnomAD v.4	0	5 htz, 0 hmz (0.000003422)		0	0	0	0	0	108 htz, 1 hmz (0.0000739)	5,965 htz, 21 hmz (0.003696)	25 htz, 0 hmz (0.00001710)	0		0		3 htz, 0 hmz (0.000001859)				6 htz, 0 hmz (0.0000041)
Other allele	KDM3A: c.1639C>T (p.Arg547Ter) (de novo, htz)	N/A		TRAK2: c.1675C>T (p.Gln559Ter) (hmz)	N/A	N/A	N/A	N/A	ASTN2: 9:116643292_116657470del (htz)	N/A	N/A	N/A		N/A		N/A				N/A

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N/A, not applicable; DL, donor loss; AL, acceptor loss; htz, heterozygous; hmz, homozygous; AOH, absence of heterozygosity; MPV, multilocus pathogenic variation.

Phenotypic information for each member of the cohort is detailed in Tables 2 and 3. Clinically, all individuals had developmental delay and/or intellectual disability. The degree of developmental delay ranged from mild to severe and included both motor and verbal delays. At last examination (average age = 5.5 years, range: 11 months–12 years), 8/18 individuals were unable to ambulate independently, and 7/18 were nonverbal. 9/18 subjects had autism or autistic-like features, and 4/18 had a diagnosis of ADHD. Another common feature was seizures and/or epileptiform activity on EEG, which were present in 10/18 individuals, with multiple individuals requiring two or more antiseizure medications for seizure control. Seizure semiologies included generalized tonic-clonic and infantile spasms, though not all seizure types were described in detail. While this manuscript was in preparation, an additional report of a child with bi-allelic LoF ASTN1 variants (c.1549C>T [p.Arg517Ter]) was published.³³ This child presented with developmental and epileptic encephalopathy (DEE), hyperreflexia, axial hypotonia, spasticity, significant developmental delays, and cerebral atrophy and thinning of the corpus callosum on MRI.³³ Taken together, these findings demonstrate that loss of ASTN1 predisposes to epileptogenesis and causes a broad spectrum of epilepsies.

Table 2.

Phenotypic summary of individuals with ASTN1-related NDDs for families 1–7

Family	F1	F2		F3	F4	F5	F6	F7
Individual	P1	P2	P3	P4	P5	P6	P7	P8
Sex	M	F	M	M	M	M	M	M
Age at last exam	3 years	5 years, 8 mo	2 years, 8 mo	3 years, 8mo	6 years	20 mo	6 years, 11 mo	9 years, 4 mo
Nucleotide	c.1523+1G>T	c.1132C>T	c.1132C>T	c.1736+5G>A	c.1599−14G>A	c.1270+1G>A	c.838_839delGA (htz); c.3125delAinsGACCACAAGTG (htz)	c.2773A>G (htz)
Protein	p.?	p.Arg378Ter	p.Arg378Ter	p.?	p.?	p.?	p.Glu280LysfsTer11; p.Glu1042GlyfsTer28	p.Met925Val
Genetic model	homozygous (tri-allelic MPV)	homozygous	homozygous	double homozygous due to distributive AOH MPV	homozygous	homozygous	compound heterozygous	double-heterozygous MPV
Other allele	KDM3A: c.1639C>T (p.Arg547Ter) (de novo, htz)	N/A	N/A	TRAK2: c.1675C>T (p.Gln559Ter) (hmz)	N/A	N/A	N/A	ASTN2: 9:116643292_116657470del (htz)
Global developmental delay/intellectual disability	Y	Y	Y	Y	Y	Y	Y	Y
Developmental milestone: walking independently	Y (2 years)	Y (3 years)	N	N	Y	N	N	Y (7 years)
Developmental milestone: verbal	N	Y	N	N	Y	Y	N	N
Developmental milestone: age at first words	N/A	4 years	N/A	N/A	3 years	18 mo	N/A	N/A
Description of verbal language abilities	NR	single-syllable words	N/A	N/A	using 2-word sentences	2–3 words	N/A	N/A
Autism/autism-like behaviors	Y	Y	Y	Y	Y	NR	Y	NR
ADHD	NR	N	N	N	Y	NR	Y	NR
Microcephaly (Z score if known)	N	Y (−2.0)	N (−0.8)	N	N	N (−0.2)	N	NR
Macrocephaly (Z score if known)	N	N	N	N (+0.2 SD)	Y (+2.0)	N	N	NR
Dysmorphic facial features (nonspecific)	NR	Y	Y	Y	Y	Y (hypertelerosim, micrognatia, frontal bossing, upslanted palpebral fissures)	Y	Y
Hypotonia	Y	Y	Y	Y	Y	Y	Y	NR
Ataxia	Y	Y	N	N	Y	N	N	N
Seizure/abnormal EEG	Y (infantile spasms, epileptiform EEG)	Y (GTC, tonic)	Y (abnormal EEG, GTC with fever)	N	Y (seizures, epileptiform EEG, well controlled on 2ASMs)	N	Y (abnormal EEG, spasms and atonic seizures in infancy, refractory clonic seizures)	Y (epilepsy on 3 ASMs)
MRI brain findings	normal	prominent cerebellar vermian folia, thinCC	prominent cerebellar vermian folia, thin CC	mild cortical atrophy, ventriculomegaly, thin CC, prominent cerebellar vermian folia	normal	bilateral periventricular hyperintensities, widening frontotemporal and anterior extra-axial CSF distances	normal	cerebellar hypoplasia, agenesis of CC, hydrocephalus, lissencephaly, colpocephaly
Other neurologic exam findings	NR	hyperreflexia	hyperreflexia	hyperreflexia	difficulty climbing stairs and jumping, flat footed	hyperreflexia, strabismus	sleeping disorder	blurry vision, strabismus, hydrocephalus

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N/A, not applicable; NR, not reported; N, no; Y, yes; htz, heterozygous; hmz, homozygous; AOH, absence of heterozygosity; MPV, multilocus pathogenic variation; DEE, developmental and epileptic encephalopathy; CSF, cerebrospinal fluid; GTC, generalized tonic clonic; ASM, antiseizure medication; CC, corpus callosum.

Table 3.

Phenotypic summary of individuals with ASTN1-related NDDs for families 8–11 and a previously published individual

Family	F8	F9		F10		F11				Ayaz et al.³³
Individual	P9	P10	P11	P12	P13	P14	P15	P16	P17	N/A
Sex	M	F	F	F	M	M	F	M	F	F
Age at last exam	12 years	4 years	3 years	NR	7 years	10 years	NR	NR	NR	2.5 years
Nucleotide	c.3283A>C (htz), c.2770C>T (htz)	c.2224G>C	c.2224G>C	c.3159_3160del	c.3159_3160del	c.3334C>T	c.3334C>T	c.3334C>T	c.3334C>T	c.1549C>T
Protein	p.Met1095Leu, p.His924Tyr	p.Gly742Arg	p.Gly742Arg	p.Gln1053HisfsTer13	p.Gln1053HisfsTer13	p.Arg1112Ter	p.Arg1112Ter	p.Arg1112Ter	p.Arg1112Ter	p.Arg517Ter
Genetic model	compound heterozygous	homozygous	homozygous	homozygous	homozygous	homozygous	homozygous	homozygous	homozygous	homozygous
Other allele	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A
Global developmental delay/intellectual disability	Y	Y	Y	Y	Y	Y	Y	Y	Y	Y
Developmental milestone: walking independently	Y	N	N	Y (3 years)	Y (2.5 years)	N	NR	NR	NR	N
Developmental milestone: verbal	Y	NR	NR	Y	Y	N	NR	NR	NR	N
Developmental milestone: age at first words	7 years	NR	NR	3 years	1.5 years	N/A	NR	NR	NR	N/A
Description of verbal language abilities	NR	NR	NR	abnormal prosody and content	abnormal content	N/A	NR	NR	NR	N/A
Autism/autism-like behaviors	NR	NR	NR	Y	Y	Y	NR	NR	NR	NR
ADHD	NR	NR	NR	NR	Y	Y	NR	NR	NR	NR
Microcephaly (Z score if known)	NR	NR	NR	NR	NR	N	NR	NR	NR	N
Macrocephaly (Z score if known)	NR	NR	NR	N/A	NR	N	NR	NR	NR	N
Dysmorphic facial features (nonspecific)	Y	Y	Y	N	N	Y	NR	NR	NR	NR
Hypotonia	NR	NR	NR	Y	Y	Y	NR	NR	NR	Y (axial)
Ataxia	NR	NR	NR	Y	N	NR	NR	NR	NR	N
Seizure/abnormal EEG	Y (seizures, on multiple ASMs)	NR	NR	N	Y (epileptiform EEG)	Y (seizures)	NR	NR	NR	Y (DEE)
MRI brain findings	polymicrogyria	thin CC	thin CC	cerebellar hypoplasia	normal	malformed hippocampus	NR	NR	NR	diffuse cerebral atrophy, thin CC
Other neurologic exam findings	spastic tetraplegia	NR	NR	decreased pain sensitivity	decreased pain sensitivity	psychomotor delay	NR	NR	NR	hyperreflexia, appendicular hypertonia, spasticity, ophthalmoparesis

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Subtle dysmorphic facial features were appreciated in 11/18 subjects. Though individual 6 (II-1 in Figure 1) was described as having hypertelorism, micrognathia, frontal bossing, and upslanted palpebral fissures, other facial dysmorphisms were regarded as minor and nonspecific, with no recognizable pattern of human malformation or facial gestalt yet emerging. Individual 9 (II-1 in Figure 1) demonstrates facial features distinct from those of other individuals for whom photographs are available (Figure 2). It is possible that this individual carries other variants that could modify the facial phenotype that escaped ES. Other neurological abnormalities detected in a subset of subjects included hypotonia (10/18), ataxia (4/18), hyperreflexia (5/18), decreased pain sensitivity (2/18), spastic tetraplegia (2/18), psychomotor retardation (1/18), and ophthalmoparesis (1/18). Though head circumference was within the normal range for most when available, one individual had microcephaly, and one had macrocephaly.

Brain malformations and dysmorphic facial features in individuals with *ASTN1* variants

MRI results obtained clinically were available for some of the individuals, demonstrating thinning of the corpus callosum (green arrow), cerebellar dysgenesis (orange arrow), cerebral atrophy (light blue arrow), hydrocephalus (black arrow), lissencephaly (yellow arrow), colpocephaly (dark blue arrow), polymicrogyria (purple arrow), and malformation of hippocampus (pink arrow). Individual photographs demonstrate nonspecific dysmorphic facial features.

Fifteen subjects underwent brain MRI. Brain imaging was normal in 4/15 individuals and abnormal in 11/15 individuals. Brain malformations appreciated were varied and included mild cerebral atrophy, polymicrogyria, lissencephaly, colpocephaly, malformation of the hippocampus, bilateral periventricular hyperintensities, and dysgenesis of the corpus callosum and cerebellum. The most common brain malformations observed in this cohort are callosal dysgenesis (7/15) and dysgenesis of the cerebellum (5/15) (Figure 2).

Molecular findings of variants in ASTN1

ASTN1 (GenBank: NM_004319.3) is located in chromosomal region 1q25.2 and contains 23 exons. We identified 17 individuals from 11 unrelated families with ASTN1 variants and additionally included one individual from a recently reported family³³ (Figure 1). Variant details are shown in Table 1. Among the 18 individuals, there are a total of 14 different variants, including four missense variants, eight predicted LoF (pLoF) variants, and two near-splice-site variants (c.1736+5G>A [p.?] and c.1599−14G>A [p.?]). The pLoF variants identified in the homozygous state include c.1523+1G>T (p.?) (intron 8 of 22), c.1132C>T (p.Arg378Ter) (exon 6 of 23), c.1270+1G>A (p.?) (intron 6 of 22), c.3159_3160del (p.Gln1053HisfsTer13) (exon 19 of 23), c.3334C>T (p.Arg1112Ter) (exon 20 of 23), and c.1549C>T (p.Arg517Ter) (exon 9 of 23). One individual (individual 7, II-1 in Figure 1) had two pLoF variants in the compound heterozygous state: c.838_839delGA (p.Glu280LysfsTer11) (exon 3 of 23) and c.3125delAinsGACCACAAGTG (p.Glu1042GlyfsTer28) (exon 19 of 23). The ASTN1 nonsense and frameshift variants are predicted to undergo nonsense-mediated decay (NMD) and therefore likely represent null alleles. The canonical splice site variants c.1523+1G>T and c.1270+1G>A, as well as the near-splice-site variants c.1736+5G>A and c.1599−14G>A, were predicted to disrupt splicing by SpliceAI (Table 1).

All identified ASTN1 variants are rare to ultra-rare in gnomAD v.4.1.0. Nearly all are absent in the homozygous state in gnomAD v.4.1.0 except for c.2773A>G (p.Met925Val) (one homozygote, allele frequency = 0.0000739) and c.3283A>C (p.Met1095Leu) (21 homozygotes, allele frequency = 0.003696). Both variants are seen in the heterozygous state in our cohort in individuals who have additional variants that are hypothesized to interact with these more common variants to confer pathogenicity. The heterozygous variant c.2773A>G (p.Met925Val) was seen in individual 8 (II-1 in Figure 1), who also has a heterozygous deletion in ASTN2 (116643292_116657470del); the heterozygous variant c.3283A>C (p.Met1095Leu) was seen in individual 9 (II-1 in Figure 1), who is compound heterozygous for ASTN1 c.2770C>T (p.His924Tyr). ASTN1 c.2770C>T (p.His924Tyr) is absent in the homozygous state in gnomAD v.4.1.0 (allele frequency = 0.00001710). Thus, c.2773A>G (p.Met925Val) and c.3283A>C (p.Met1095Leu) may act as hypomorphic alleles, which only cause disease in combination with a second, more damaging allele. This phenomenon has been previously described, such as in the case of parkinsonism-dystonia 3, childhood onset (PKDYS3, MIM: 619738), an NDD that presents with infantile- or childhood-onset progressive movement disorders. PKDYS3 is caused by the compound inheritance of one ultra-rare pathogenic WARS2 (MIM: 604733, GenBank: NM_015836.4) LoF variant and the hypomorphic WARS2 variant c.37T>G (p.Trp13Gly) (gnomAD v.4.1.0: 22 homozygotes, allele frequency = 0.003588). WARS2 p.Trp13Gly homozygotes are asymptomatic, whereas individuals with bi-allelic WARS2 LoF variants have a more severe mitochondrial neurodevelopmental phenotype (MIM: 617710).⁴² Most missense variants were predicted to be damaging by the in silico pathogenicity predictor Combined Annotation Dependent Depletion (CADD) (Table 1).

Mini-gene splicing assays demonstrate splicing abnormalities secondary to ASTN1 variants

This study includes two canonical splice-site variants (c.1523+1G>T and c.1270+1G>A) and two near-splice-site variants (c.1736+5G>A and c.1599−14G>A). In addition to the expected disruption of splicing seen with the in silico predictor SpliceAI, we sought to increase confidence in these predictions, particularly for the near-splice-site variants. A mini-gene splicing assay was performed, which was in concordance with the predicted disruption of splicing for all splice-site and near-splice-site variants in this cohort. For canonical splice-site variant c.1523+1G>T and the near-splice-site variant c.1736+5G>A, this assay demonstrated exon skipping (Figure 3). Exon skipping was likewise seen in the near-splice-site variant c.1599−14G>A, with exon skipping found in 27% of next-generation sequencing reads, while a truncated exon was identified in 65% of the reads, and normal splicing was present in the remaining 8%. For the canonical splice-site variant c.1270+1G>A, this assay demonstrated intron retention. These results provide functional evidence supporting the pathogenic impact of the identified splice-site and near-splice-site variants.

Mini-gene splicing assay demonstrates splicing defects secondary to *ASTN1* single-nucleotide variants

(A) Mini-gene splicing assay strategy for *ASTN1* variants. The exon of interest (black) including flanking *ASTN1* intronic sequences (gray) were cloned into the mini-gene construct within *SMN1* introns 7 and 8 (orange). GFP is shown in green.

(B) Results of RT-PCR performed on either reference sequence (indicated with REF) or *ASTN1_*c.1523+1G>T variant sequence. The reference sequence demonstrates a band with the inclusion of exon 8. The variant sequence demonstrates a lower-molecular-weight band. Sanger sequencing results are shown for the REF exon 8 as well as dominant, lower-molecular-weight RT-PCR bands for the *ASTN1*_c.1523+1G>T variant sequence (VAR), which demonstrates exon 8 skipping.

(C) Results of RT-PCR performed on either reference sequence (indicated with REF) or *ASTN1_*c.1270+1G variant sequence. The reference sequence demonstrates a band with the inclusion of exon 6. The variant sequence demonstrates two higher-molecular-weight bands. Sanger sequencing results are shown for the REF exon 6 as well as higher-molecular-weight RT-PCR bands for the *ASTN1_*c.1270+1G variant sequence (VAR), which demonstrates intron 6 inclusion (blue arrow) and intron inclusion in addition to inclusion of *SMN1* intron 7 from the assay construct (orange arrow).

(D) Results of RT-PCR performed on either reference sequence (indicated with REF) or *ASTN1_*c.1736+5G>A variant sequence. The reference sequence demonstrates a band with the inclusion of exon 10. The variant sequence demonstrates a lower-molecular-weight band. Sanger sequencing results are shown for the REF exon 10 as well as lower-molecular-weight RT-PCR bands for the *ASTN1_*c.1736+5G>A variant sequence (VAR), which demonstrates exon 10 skipping.

(E) Results of RT-PCR performed on either reference sequence (indicated with REF) or *ASTN1_*c.1599−14G>A variant sequence. The reference sequence demonstrates a band with the inclusion of exon 10. The variant sequence demonstrates three bands, one of the same weight as the reference sequence and two lower-molecular-weight bands. Sanger sequencing results are shown for the REF exon 10 as well as all three RT-PCR bands for the *ASTN1_*c.1599−14G>A variant sequence (VAR), which demonstrate the full exon (orange arrow), a truncated exon (green arrow), and exon 10 skipping (blue arrow). These findings are also confirmed with amplicon sequencing.

Developmental brain expression of ASTN1

ASTN1 and ASTN2 are expressed in the developing brain and work together to regulate neuronal migration in laminar structures (Figure 4A). To better understand the role of ASTN1 and ASTN2 in the developing human brain, we examined transcriptomic data from the Human Brain Atlas and a second-trimester human fetal brain single-cell RNA-sequencing dataset.⁴³^,⁴⁴ Interestingly, they are both expressed in Cajal-Retzius cells,⁴⁴ which play a critical role in the laminar organization of the cortex and hippocampus (Figures 4B and 4C).⁴⁵ This may suggest additional roles for these genes, with ASTN1 potentially serving as a ligand drawing neurons from the radial glial process into their ultimate laminar positions. From prenatal development to adulthood, ASTN1 and ASTN2 are robustly expressed and follow similar expression patterns to each other, as well as to other common NDD genes (Figure 4D).⁴⁶ Expression patterns of ASTN1, ASTN2, and other genes involved in neuronal migration,⁴⁷ such as RELN (MIM: 600514), DCX (MIM: 300121), FLNA (MIM: 300017), CDK5 (MIM: 123831), and VLDLR (MIM: 192977), are also comparable (Figure 4E), although DCX shows elevated expression through 24 weeks post-conception before declining to lower adult levels by 2 years of age (Figure 4E).

*ASTN1* developmental expression

(A) Schematic of ASTN1 and ASTN2 roles in neuronal migration.

(B) Single-cell RNA-seq expression of *ASTN1* in the second-trimester human brain as visualized in NeuroTri2-VISDOT.⁴⁴

(C) Single-cell RNA-seq expression of *ASTN2* in the second-trimester human brain as visualized in NeuroTri2-VISDOT.⁴⁴

(D) Pre- and postnatal expression averaged over various brain regions of *ASTN1* and *ASTN2* compared to known NDD genes from the BrainSpan Atlas of the Developing Human Brain.⁴³

(E) Pre- and postnatal expression averaged over various brain regions of *ASTN1* and *ASTN2* compared to disease-causing genes with roles in neuronal migration from the BrainSpan Atlas of the Developing Human Brain.⁴³

Evidence for MPV and suggested genetic models

In addition to the ASTN1 variants described above, ES analysis identified three distinctive genetic models of MPV in families within our cohort: double heterozygous, tri-allelic, and double homozygous due to distributive AOH. Double-heterozygous MPV was previously characterized in individual 8 (II-1 in Figure 1), who has a heterozygous variant in ASTN1 (c.2773A>G [p.Met925Val]) in addition to a heterozygous 14.178 kb deletion including exon 17 in ASTN2 (116643292_116657470del).³¹ Importantly, ASTN1 and ASTN2 interact during neurogenesis to enable radial migration of neurons along radial glia. Along with the homozygous ASTN1 variant c.1523+1G>T, individual 1 (II-1 in Figure 1) has a de novo heterozygous pLoF KDM3A (MIM: 611512; GenBank: NM_018433.6) variant (c.1639C>T [p.Arg547Ter]), which is ultra-rare (gnomAD v.4.1.0 allele frequency = 0.000002052), is predicted to be damaging (CADD 36), and should trigger NMD. KDM3A pLoF has been reported to be associated with autism.⁴⁸^,⁴⁹^,⁵⁰ KDM3A shows a high level of constraint against pLoF variants in population databases (gnomAD v.2.1.1: pLI = 1 and LOEUF = 0.22; gnomAD v.4.1.0: pLI = 0.99 and LOEUF = 0.47), comparable to highly penetrant haploinsufficient NDD disease genes and moderate-risk autism spectrum disorder (ASD) genes⁵¹ (Table S1). This individual exemplifies tri-allelic MPV with three pathogenic alleles at two loci. Individual 4 (II-3 in Figure 1) demonstrates double-homozygous MPV secondary to distributive AOH with homozygous variants in ASTN1 (c.1736+5G>A) and TRAK2 (MIM: 607334; GenBank: NM_015049.3) (c.1675C>T [p.Gln559Ter]). The TRAK2 variant is absent in gnomAD v.4.1.0, has a CADD score of 40, and is predicted to undergo NMD. TRAK2 is not presently linked to human disease but is a strong candidate NDD gene for several reasons. First, TRAK2 is involved in mitochondrial and GABA-A receptor trafficking.⁵²^,⁵³^,⁵⁴ Second, Trak2^−/− mice are viable and exhibit a decreased anxiety-related response, abnormal behavior, and decreased thigmotaxis.⁵⁵ Finally, the TRAK2 paralog TRAK1 (MIM: 608112) is linked to DEE68 (MIM: 618201).⁵⁶ There are three homozygous TRAK2 pLoF variants in gnomAD v.2.1.1 and gnomAD v.4.1.0: c.2596C>T (p.Gln866Ter), c.2678_2700del (p.Ile893SerfsTer20), and c.682C>T (p.Arg228Ter) (Table S2). TRAK2 p.Gln688Ter and p.Ile893SerfsTer20 both fall within the final exon of TRAK2 and are predicted to escape NMD. Both variants should therefore result in a minor truncation of the 914 amino acid protein in a region lacking annotated functional domains. Consequently, both are less likely to represent true LoF alleles. TRAK2 p.Arg228Ter falls within exon 6 of 16 and thus should be an NMD-triggering LoF allele. A single TRAK2 p.Arg228Ter homozygote is found in gnomAD v.4.1.0 but not in either gnomAD v.4.1.0 (non-UK Biobank [UKB]) or gnomAD v2.1.1. As individuals with rare protein-truncating and -damaging missense variants in monogenic developmental disorder genes have been identified in the UKB, the presence of a single TRAK2 pLoF homozygote in the UKB does not exclude the association of TRAK2 with autosomal-recessive NDDs.⁵⁷^,⁵⁸ The total AOH for this individual was 497.6 Mb, consistent with a coefficient of consanguinity of 0.171. ES studies of individual 4 document that both genes, ASTN1 on chromosome 1 and TRAK2 on chromosome 2, map within an AOH interval consistent with identity by descent versus identity by state (Figure 1).

Discussion

The role of ASTN1 in neurodevelopment

This allelic series defines the human phenotypic characteristics of ASTN1-related NDDs, which are likely the result of the disruption of the radial migration of neurons during neurogenesis secondary to impaired neuron-glia adhesion. ASTN1 is localized on postnatal neurons and acts as a neuronal-glial ligand at the migrational junction.³^,⁴^,⁵^,⁶^,⁷^,⁸^,⁹^,¹⁰ ASTN1-mediated adhesion is necessary for appropriate radial migration of neurons along radial glial cells, with ASTN1 blockade resulting in delayed migration and Astn1-knockout mice demonstrating impaired neuronal migration, abnormal cerebellar Purkinje cell development, and impaired balance and coordination.¹² However, despite this critical role of ASTN1 in neurodevelopment, mice lacking Astn1 have minimal gross brain malformations, with only an approximately 10% decrease in cerebellar area.¹² With this in mind, it is perhaps not surprising that some individuals with pathogenic variants in ASTN1 do not have brain malformations visible at the resolution of an MRI and, likewise, that there is not a significant alteration in head circumference found in most individuals. In fact, the most common brain malformation identified is thinning of the corpus callosum, which can be the result of impaired development of the cerebral cortical layers, followed by cerebellar dysgenesis. These findings in human subjects are thus consistent with those observed in the Astn1-knockout mice.

This study demonstrates impaired motor development and ataxia, which would be expected and consistent with the Astn1 knockout mice with impaired cerebellar development. Importantly, the role of the cerebellum in other non-motor neurodevelopmental processes is increasingly appreciated.¹³ In addition to its role in motor development, the cerebellum is known to be important for cognition, affect, and social cognition, with developmental alterations in cerebellar structure and function implicated in several NDDs, including autism and ADHD.⁵⁹^,⁶⁰ In individuals with autism, the migration and size of cerebellar Purkinje cells are altered,⁶¹ and a decreased size of the vermis is observed.⁶² Among the individuals in this study, impaired language development, autism, and ADHD were prevalent, which may indeed be secondary to impaired cerebellar development. However, these features, in addition to other characteristics of this cohort, including epilepsy and hypotonia, may be related to cortical dysgenesis as well, given the role of ASTN1 in cortical development.

Interestingly, individual 8 (II-1 in Figure 1) has the most significant brain malformations and is the only individual with variants in both ASTN1 and ASTN2, which interact to promote normal neuronal migration, with ASTN2 regulating the cell surface expression of ASTN1 during neuronal radial migration.¹¹ Heterozygous ASTN2 deletions, like that seen in this individual, have long been associated with a range of NDDs,¹⁸^,¹⁹^,²⁰^,²¹^,²²^,²³^,²⁴^,²⁵^,²⁶^,²⁷ and Astn2-knockout mice have alterations in cerebellar circuitry¹⁴ and autism-like behaviors.¹⁵ Furthermore, loss of ASTN2 has been shown to significantly upregulate ASTN1 expression in human neuronal cells⁶³ and mouse cerebellum.¹⁵ While limited phenotypic details are available regarding individuals with heterozygous ASTN2 deletions, it is apparent that only a subset have brain malformations. We hypothesize that disruption in both ASTN1 and ASTN2 results in more significant impairment of neuronal migration, an epistatic interaction leading to a more severe clinical phenotype and more prominent brain malformations. This may explain the highly heterogeneous phenotypes previously associated with ASTN2 deletions, with only a subset of individuals having brain malformations. Further study of individuals with ASTN2 deletions is needed to test this hypothesis. Similarly, polymicrogyria was noted in only individual 9 (II-1 in Figure 1). As the extent and distribution of polymicrogyria vary considerably and its appearance grows more prominent as myelination progresses, it is possible that we have underestimated the prevalence of polymicrogyria in ASTN1-related NDD.⁶⁴

The role of MPV in ASTN1-related NDDs

MPV, the occurrence of pathogenic variant alleles in two or more loci, is increasingly recognized in consanguineous and non-consanguineous populations in clinical and research settings.³¹^,⁶⁵^,⁶⁶^,⁶⁷ Recognition of MPV is critical, as it can shape blended phenotypes and contribute to phenotypic severity and intrafamilial variability. Consanguinity increases the probability of MPV in a population, which can be due to larger regions of AOH in individuals born to consanguineous families.³¹^,⁴¹ This study of research subjects with pathogenic variant alleles in ASTN1 demonstrates three genetic models of MPV, including tri-allelic, double heterozygous, and double homozygous due to distributive AOH, and underscores the contribution of MPV to the genetic origin of recessive NDDs.

Tri-allelic MPV is demonstrated with homozygous variants in ASTN1 and a de novo heterozygous LoF variant in a candidate gene, KDM3A—three variant alleles at two loci. KDM3A has been reported as a candidate gene in several individuals, including a single exon deletion in an individual with severe intellectual disability, autism, and epilepsy⁴⁸; a de novo missense variant in an individual with autism and severe developmental disorder⁴⁹; and a de novo stop-gain in an individual in an autism cohort.⁵⁰ Though limited phenotypic information is available regarding these individuals with candidate KDM3A variant alleles, individual 1 (II-1 in Figure 1) in the current study has a de novo pLoF KDM3A variant and has features not previously associated with this gene, such as impaired coordination. We hypothesize that the combination of variants in ASTN1 and KDM3A in this individual with tri-allelic MPV results in this blended phenotype. Alternatively, KDM3A LoF may be associated with moderate ASD risk with reduced penetrance, like ITSN1 (MIM: 602442), NAV3 (MIM: 611629), SCAF1 (MIM: 617264), and HNRNPUL2.⁵¹ If KDM3A LoF confers moderate ASD risk, the phenotypic consequence of the de novo KDM3A variant may be negligible. Indeed, our work suggests that individual 1’s phenotype could be entirely within the spectrum of ASTN1-related NDD.

Double-heterozygous MPV is observed by the compound inheritance of heterozygous variants in both ASTN1 and ASTN2.³¹ While the heterozygous variant in ASTN1 in this individual is more frequent than might be expected for a pathogenic allele and has a CADD score of 12.77, we hypothesize that the individual harboring both of these variants has a severe phenotype due to the synergistic function of these genes in neurodevelopment and the manner in which they interact to facilitate neuronal migration. This speculation is based on the known roles and interactions between ASTN1 and ASTN2 in neurodevelopment. ASTN2 copy-number variants (CNVs) are associated with highly heterogeneous phenotypes ranging from ADHD, anxiety/mood disorders, and learning disabilities to severe NDDs and rare brain malformations. While de novo ASTN2 CNVs were identified in several individuals, the inheritance of ASTN2 CNVs from unaffected parents has also been demonstrated, thereby suggesting the reduced penetrance of ASTN2 LoF.²¹ Double-heterozygous MPV may thus explain a subset of the phenotypic variability and reduced penetrance of ASTN2 CNVs. Ultimately, further validation through additional individual identification and functional studies is needed to confirm the pathologic effect of the interaction of these variants in this individual and others.

Double- or higher-order-homozygous MPV due to distributive AOH can occur in families with consanguinity,⁶⁵ as is seen in individual 4 (II-3 in Figure 1). Here, we describe an individual with homozygous variants in both ASTN1 and TRAK2. TRAK2 is necessary for dendritic development and plays an important role in mitochondrial trafficking in dendrites of at least cortical and hippocampal neurons.⁵²^,⁵³ There is evidence that TRAK2 also serves as a GABA_A receptor-associated trafficking protein.⁵⁴ At this time, TRAK2 variants do not have a known disease association; however, knockout mice and de novo variants in the GABA_A receptor subunit associated with TRAK1, a gene encoding a trafficking protein in the same family as TRAK2, have been associated with epilepsy.⁵⁴ Given its biological function, absence in gnomAD v.4.1.0, the near-complete absence of bi-allelic TRAK2 pLoFs in gnomAD, and the CADD score of 40, we hypothesize that this homozygous TRAK2 variant is contributory. Individual 4 has developmental delay at the more severe end of the ASTN1-related NDD spectrum, being unable to walk or talk at the last exam at age 3 years and 8 months. Additionally, on brain MRI, he has cortical atrophy, which is a rare finding in ASTN1-related NDD. Thus, we hypothesize that both homozygous variants contribute to his blended clinical phenotype, resulting in double-homozygous MPV.

Individuals with MPV have been included in this series to highlight both this increasingly appreciated phenomenon and the importance of consideration of multiple potential diagnoses when evaluating individuals with NDDs. Identifying additional disease-causing variants can have repercussions for treatment and family counseling, whether the additional allele results in a blended phenotype or an overlapping phenotype, in which it is clinically difficult to weigh the relative contributions of each genetic diagnosis. When counseling families in such cases, it is important to discuss how these findings may factor into intrafamilial variability and recurrence risk, which may include uncertainty regarding the contribution of each variant to the clinical phenotype.

Conclusions

This study provides a thorough phenotypic description of ASTN1-related NDDs and thereby strengthens the gene-disease associations of ASTN1, ASTN2, KDM3A, and TRAK2. We demonstrate that bi-allelic LoF variation in ASTN1 causes mild to severe developmental delay or intellectual disability and is frequently associated with autism, ADHD, and/or epilepsy. Other recurrent abnormalities include hypotonia, nonspecific dysmorphic facial features, hyperreflexia, and ataxia. We hypothesize that ASTN1-related NDDs result from disrupted neuronal migration in the developing brain, particularly in the cerebellum and cerebral cortex, where ASTN1 mediates radial migration. Additionally, our study highlights the importance of considering MPV when evaluating for genetic diagnoses of NDDs, especially in consanguineous families.

Data and code availability

All data described in this study are provided within the article and supplemental information. Raw sequencing data and deidentified clinical data are available from the corresponding authors upon request. All ES and GS data generated by the BHCMG/BCM-GREGoR for which informed consent for deposition into controlled-access databases was provided were deposited into either dbGaP or the AnVIL repository under the study name Baylor-Hopkins Center for Mendelian Genomics or the GREGoR consortium (https://anvilproject.org/). The accession number for the data reported in this paper is BHCMG dbGaP: phs000711.v7.p2.

Acknowledgments

The authors would like to acknowledge the families who participated in the study. The data in this manuscript were previously presented at the annual meetings of the American Society of Human Genetics (2024) and the Genomics Research to Elucidate the Genetics of Rare Disease (GREGoR) Consortium (2024). This study was supported in part by a grant from the US National Human Genome Research Institute (NHGRI) and the National Heart, Lung, and Blood Institute (NHBLI) to the Baylor-Hopkins Center for Mendelian Genomics (BHCMG, UM1 HG006542, J.R.L.); an NHGRI grant as part of the GREGoR Consortium (U01 HG011758 to J.E.P., J.R.L., and R.A.G.); an NHGRI grant to the Baylor College of Medicine Human Genome Sequencing Center (U54HG003273 to R.A.G.); and the US National Institute of Neurological Disorders and Stroke (NINDS) (R35NS105078 to J.R.L.). D.P. was supported by NINDS 1K23 NS125126-01A1 and the Rett Syndrome Research Trust fellowship award from the International Rett Syndrome Foundation (IRSF grant #3701-1). D.G.C. was supported by the National Institute of Neurological Disorders and Stroke of the National Institutes of Health under award number K12NS098482 and by an MDA Development Grant (873841). K.M.B. was supported by the GREGoR Consortium Research Grant, Research to Prevent Blindness - MEE Grant 2023, P30EY014104 (MEEI core support), and the Foundation Fighting Blindness (EGI-GE-1218-0753-UCSD). N.A. was supported by the Research, Development and Innovation Authority (RDIA), Kingdom of Saudi Arabia (12996-iau-2023-TAU-R-3-1-HW-). K.M.B. was supported by National Eye Institute (R01EY035717).

Author contributions

Conceptualization, J.M.L. and D.G.C.; data curation, J.M.L., D.G.C., H.D., A.S., J.L., T.B., J.D., E.U., M.Y.C., A.Y., H.Y.E., E.Y.G., A.M., N.A., P.G., W.W., E.K., L.A., D.P., D.M., M.S.Z., F.S.A., J.G.G., S.N.J., R.A.G., and J.E.P.; investigation, J.M.L., D.G.C., R.S., and K.M.B.; resources, J.R.L.; writing – original draft, J.M.L.; writing – review & editing, J.M.L., D.G.C., and J.R.L.; supervision, J.R.L.

Declaration of interests

The authors declare no competing interests.

Published: January 15, 2026

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ajhg.2025.12.011.

Contributor Information

Jesse M. Levine, Email: jesse.levine@bcm.edu.

James R. Lupski, Email: jlupski@bcm.edu.

Web resources

Baylor College of Medicine Human Genome Sequencing Center, https://www.hgsc.bcm.edu
Baylor College of Medicine Lupski Lab, https://github.com/BCM-Lupskilab
BrainSpan, https://www.brainspan.org/
CADD, https://cadd.gs.washington.edu/
GenBank, https://www.ncbi.nlm.nih.gov/genbank/
gnomAD Browser, https://gnomad.broadinstitute.org/
Online Mendelian Inheritance in Man, http://www.omim.org
SpliceAI, https://spliceailookup.broadinstitute.org/

Supplemental information

Document S1. Figure S1 and Tables S1 and S2

mmc1.pdf^{(651.4KB, pdf)}

Document S2. Article plus supplemental information

mmc2.pdf^{(11MB, pdf)}

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Associated Data

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Supplementary Materials

Document S1. Figure S1 and Tables S1 and S2

mmc1.pdf^{(651.4KB, pdf)}

Document S2. Article plus supplemental information

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Data Availability Statement

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PERMALINK

Bi-allelic variants in neuronal adhesion molecule astrotactin 1 gene ASTN1 cause diverse neurodevelopmental disorders

Jesse M Levine

Daniel G Calame

Riccardo Sangermano

Haowei Du

Ahmed Saad

Jasmin Lisfeld

Tatjana Bierhals

Jonas Denecke

Eyyup Uctepe

Merve Yoldas Celik

Ahmet Yesilyurt

Hilal Yildiz Er

Elif Yilmaz Gulec

Aziza Mushiba

Naif Almontashiri

Pawel Gawlinski

Wojciech Wiszniewski

Ender Karaca

Lama Alabdi

Davut Pehlivan

Dana Marafi

Maha S Zaki

Fowzan S Alkuraya

Joseph G Gleeson

Shalini N Jhangiani

Richard A Gibbs

Jennifer E Posey

Kinga M Bujakowska

James R Lupski

Summary

Introduction

Subjects, material, and methods

Participant ascertainment and identification

Genomic sequencing and variant prioritization

Functional mini-gene splicing assay

AOH calculations

Results

Identification and phenotypic characterization of individuals with NDDs likely due to ASTN1 variants

Figure 1.

Table 1.

Table 2.

Table 3.

Figure 2.

Molecular findings of variants in ASTN1

Mini-gene splicing assays demonstrate splicing abnormalities secondary to ASTN1 variants

Figure 3.

Developmental brain expression of ASTN1

Figure 4.

Evidence for MPV and suggested genetic models

Discussion

The role of ASTN1 in neurodevelopment

The role of MPV in ASTN1-related NDDs

Conclusions

Data and code availability

Acknowledgments

Author contributions

Declaration of interests

Footnotes

Contributor Information

Web resources

Supplemental information

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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