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. Author manuscript; available in PMC: 2025 Apr 1.
Published in final edited form as: Genet Med. 2023 Dec 27;26(4):101057. doi: 10.1016/j.gim.2023.101057

Biallelic loss-of-function variants in CACHD1 cause a novel neurodevelopmental syndrome with facial dysmorphism and multisystem congenital abnormalities

Marcello Scala 1,2,3,23, Kamal Khan 4,23, Claire Beneteau 5,6,7,23, Rachel G Fox 8, Sandra von Hardenberg 9, Ayaz Khan 4, Madeleine Joubert 6,10, Lorraine Fievet 11, Marie Musquer 6,10, Claudine Le Vaillant 12, Julie Korda Holsclaw 11, Derek Lim 13,14, Ann-Cathrine Berking 9, Andrea Accogli 1, Thea Giacomini 1,15, Lino Nobili 1,15, Pasquale Striano 1,2, Federico Zara 1,3, Annalaura Torella 16,17, Vincenzo Nigro 16,17, Benjamin Cogné 5,18, Max R Salick 19, Ajamete Kaykas 19, Kevin Eggan 20, Valeria Capra 3, Stéphane Bézieau 5,18, Erica E Davis 4,21,24,*, Michael F Wells 8,20,22,24,*
PMCID: PMC11910193  NIHMSID: NIHMS2059687  PMID: 38158856

Abstract

Purpose:

We established the genetic etiology of a syndromic neurodevelopmental condition characterized by variable cognitive impairment, recognizable facial dysmorphism, and a constellation of extra-neurological manifestations.

Methods:

We performed phenotypic characterization of six participants from four unrelated families presenting with a neurodevelopmental syndrome and used exome sequencing (ES) to investigate the underlying genetic cause. To probe relevance to the neurodevelopmental phenotype and craniofacial dysmorphism, we established two- and three-dimensional human stem cell-derived neural models, and generated a stable cachd1 zebrafish mutant on a transgenic cartilage reporter line.

Results:

Affected individuals showed mild cognitive impairment, dysmorphism featuring oculo-auriculo abnormalities, and developmental defects involving genitourinary and digestive tracts. ES revealed biallelic putative loss-of-function variants in CACHD1 segregating with disease in all pedigrees. RNA sequencing in CACHD1-depleted neural progenitors (NPCs) revealed abnormal expression of genes with key roles in Wnt signaling, neurodevelopment, and organ morphogenesis. CACHD1 depletion in NPCs resulted in reduced percentages of post-mitotic neurons and enlargement of 3D neurospheres. Homozygous cachd1 mutant larvae showed mandibular patterning defects mimicking human facial dysmorphism.

Conclusion:

Our findings support the role of loss-of-function variants in CACHD1 as the cause of a rare neurodevelopmental syndrome with facial dysmorphism and multisystem abnormalities.

INTRODUCTION

Voltage-gated calcium channels (VGCCs) act as electrical excitability transducers in neuronal, cardiac, and muscle cells by mediating Ca2+ influx in response to action potentials and subthreshold depolarizing signals1. Distinct molecular subtypes of VGCCs are implicated in signal transduction in different cell types1. This functional specialization reflects specific physiological, pharmacological, and regulatory properties of VGCC subtypes that play critical roles in hormone secretion, transmitter release, and both excitation-transcription and excitation-contraction coupling1. Dysfunctional VGCCs increase the risk for schizophrenia, heart arrhythmias, muscle weakness, and a broad range of other disorders known as channelopathies2. The physiological properties of VGCCs—and the cells in which they are expressed—are largely determined by the pore-forming α1 subunits, while α1-interacting auxiliary β and α2δ subunits modulate cell-surface expression and trafficking3,4. The importance of these subunits on VGCC physiology is well-established, but it is less clear if other subunit-interacting proteins can influence channel function and contribute to the onset of disease.

The α2δ-like cache domain containing 1 (CACHD1; MIM: 620144) protein forms a complex with and modulates the activity and expression of high voltage-activated N-type (Cav2.2) and low voltage-activated T-type (Cav3) channels5,6. High levels of CACHD1 mRNA and protein are present in the mammalian central nervous system (CNS), especially in the cerebellum, hippocampus, and thalamus5. At the tissue level, CACHD1 expression overlaps the distribution of α2δ−3 proteins and Cav3 subunits; at the subcellular level, this protein colocalizes with Cav2.2 and Cav3 channels at the cell surface5,6. Overexpression of CACHD1 significantly increases Cav2.2 and Cav3 current density and maximal conductance, and CACHD1 is thought to exert these physiological effects by increasing cell surface expression and reducing endocytosis of these N- and T-type channels5,6. Importantly, members of the α2δ protein family have putative roles in development and disease independent of VGCC modulation, suggesting that the same could be true for CACHD17.

In vitro studies have nominated CACHD1 as a regulator of neural progenitor cell (NPC) proliferation and differentiation8, as well as neuronal presynaptic function, the latter of which is consistent with the known contributions of N-type channels to presynaptic neurotransmitter release9. These early reports suggest that CACHD1 plays important roles in the mammalian brain; however, the ramifications of CACHD1 gene-disrupting variants on human health are unknown.

We assembled a cohort of six affected individuals from four unrelated families who harbor biallelic putative loss-of-function variants in CACHD1 and present with a rare neurodevelopmental syndrome characterized by variable developmental delay, cognitive impairment, craniofacial dysmorphism, and a recurrent pattern of multisystem abnormalities. Accordingly, molecular and cellular characterization of CACHD1-depleted human stem cell-derived NPCs revealed disease-relevant dysregulated pathways and defects in neurogenesis. Furthermore, cachd1-mutant zebrafish models recapitulated the craniofacial abnormalities observed in human patients. Our results provide in vitro and in vivo evidence for a pathogenic role of biallelic CACHD1 variants.

MATERIALS AND METHODS

Study approval and recruitment

Six individuals from four unrelated families were recruited after clinical assessment at different research centers and hospitals (Supplemental Methods). The study cohort was assembled using gene matching platforms10 and through international collaborative efforts. Detailed phenotypic information related to prenatal and developmental history, clinical evaluations, and medical imaging were provided by the referring physicians. Informed consent was collected from parents or legal guardians for genetic investigation and publication of clinical and genetic data. Approval was obtained as mentioned in the Ethics Declaration section.

Genetic analysis

Chromosomal microarray analysis (CMA) was performed in individuals #1–4 as described11, and the detected rearrangements were interpreted according to DECIPHER. Exome sequencing (ES) was performed on genomic DNA extracted from peripheral blood leukocytes. Parent-proband ES was performed in all participants (trios in #1–4 and duos in #5 and #6) as previously described11. Additional sequencing methodology is described in the Supplemental Methods. Genetic variants were filtered for minor allele frequency (MAF) ≤ 0.01 in genomic databases (gnomAD), presence in ClinVar, conservation (Genomic Evolutionary Rate Profiling), and predicted impact on protein structure and function. Using the Ensembl Variant Effect Predictor pipeline, several in silico tools were employed to predict variant pathogenicity, including Combined Annotation Dependent Depletion (GRCh37-v1.6 version), Polyphen-2, Mutation Taster, and Splice AI, as previously reported11. Sanger sequencing was performed to confirm candidate variants and for parental segregation analysis. Candidate variants were classified according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines. All CACHD1 variants are reported according to RefSeq NM_020925.4 (GenBank NC_000001.11) and HGVS recommendations. All variants were submitted to the Leiden Open Variation Database with the following accession numbers: #0000830402, #0000830403, #0000830406, #0000830407, #0000830408, and #0000830409.

Cell culture and CRISPR

Human embryonic stem cells (hESCs) were transduced with dox-inducible Ngn2 lentivirus and split every 4–5 days in mTesR media (Supplemental Methods). Transduced hESCs were then induced to NPCs using the Stem cell-derived NGN2-accelerated Progenitor (SNaP) method8, which were cultured in bFGF/EGF-containing maintenance media. NPCs were split weekly and plated at 120,000 cells/cm2. NPCs were transduced with Cas9-lentivirus (pLX-311-Cas9 vector) followed by selection with blasticidin (10 μg/mL). NPCs were then transduced with individual lentivirus sgRNAs targeting CACHD1 before selection with puromycin (1 μg/mL) for one week. Cells were harvested for Sanger sequencing and TIDE analysis.

RNA sequencing

RNAseq libraries were prepared with the KAPA Stranded mRNA-Seq Kit (KAPA Biosciences) and sequenced on a HiSeq 3000 (Illumina) to generate single-end 50 bp reads. FASTQ files were aligned to the human reference genome version GRCh38 and processed for DEG analysis and gene functional enrichment analysis (Supplemental Methods).

Differentiation assay

NPCs were plated for 30 days in spontaneous differentiation media before immunostaining as described in the Supplemental Methods. For each well of a 96-well plate, 4–8 fluorescent images were captured using the Cytation5 multi-mode reader (BioTek Instruments). All images were processed using the CellProfiler imaging analysis software to quantify the percentage of HuCD+ neurons or SOX2+ NPCs.

SNaP-derived neurospheres

SNaPs were dissociated in accutase and plated at 18,000 cells/well of an ultra-low attachment 96-well round bottom plate (Corning, 7007) in 150 μL of SNaP maintenance media supplemented with Y-27632 (50 μM). Two days later (Day 2), 75 μL of conditioned media was removed from each well and replaced with 150 μL of fresh SNaP maintenance media supplemented with Y-27632 (50 μM). The following day (Day 3), 125 μL of conditioned media was removed from each well using the “blast” technique and replaced with 150 μL of fresh SNaP maintenance media without Y-2763212. From Day 4 onward, 150 μL of conditioned media was removed every other day from each well using the “blast” technique and replaced with 150 μL of fresh SNaP maintenance media. Neurospheres were measured using the Cytation5 multi-mode reader using the 4X bright field objective.

Single-cell RNA sequencing

10X Genomics libraries from 10 pooled neurospheres were generated and sequenced on a HiSeq 4000 (Illumina). scRNA-seq datasets were analyzed using Seurat213. Graph-based clustering approximated different cell groups, and t-stochastic neighborhood embedding (TSNE) analysis was used for 2D representation.

Generation and molecular characterization of cachd1 zebrafish mutants

All studies performed in zebrafish were approved by the Institutional Animal Care and Use Committees (IACUC; Protocols A154–18-06 and IS00016405) at Duke University Medical Center and Northwestern University, respectively. To identify the cachd1 ortholog in zebrafish, we performed a reciprocal BLAST of human CACHD1 protein (GRCh38.p13, Ensembl: ENST00000651257.2; RefSeq: NP_065976.3) against the zebrafish genome. We synthesized sgRNA using the Gene Art precision gRNA synthesis kit (Invitrogen) per manufacturer’s instructions. We estimated sgRNA efficiency using heteroduplex analysis (n=2 wildtype [WT] and n=10 injected embryos) and TOPO-TA cloning-based sequencing (n=1 WT and n=5 injected embryos; 24 clones per embryo) as described14 (Supplementary Methods). To generate stable F2 homozygous mutants (−/−), F0 mutants carrying germline cachd1 insertion-deletions (indels) were outcrossed with WT (+/+) and then F1 heterozygous mutants (+/−) were in-crossed. To assess the expression of mutant cachd1 mRNA, we generated F2 larvae from in-crosses of heterozygous parents (n=20 larvae per genotype; 2dpf) and performed quantitative (q)PCR assays.

Zebrafish phenotyping

We used the Vertebrate Automated Screening Technology (VAST; Union Biometrica) Bioimager platform to perform live automated imaging of anterior cartilage structures in −1.4col1a1:egfp larvae as described (Supplementary Methods)15. We in-crossed genotype-matched adults (either +/+ or −/−) to generate larvae for phenotyping. Fluorescent images were assessed using ImageJ (NIH) by measuring the area surrounded by ceratohyal, palatoquadrate and Meckel’s cartilage (defined as region of interest, ROI). Statistical differences were calculated using an unpaired student’s t-test. Measurements were performed with investigators masked to genotype and repeated twice.

RESULTS

CACHD1 patients show neurodevelopmental impairment and congenital multisystem defects

Six affected individuals in four unrelated families presented with a syndromic neurodevelopmental condition featuring abnormalities in multiple organ systems (Table 1; Figure 1a–d; Supplemental Table 1; Supplemental Clinical Information). Excluding the fetal cases (#5 and #6), neurological involvement was present in all participants. A global impairment of psychomotor development was reported in individuals #3 and #4, with the latter overcoming developmental delays by the age of 2 years. The degree of cognitive impairment was generally mild. Individuals #1–3 exhibited learning disabilities leading to writing and speech impairment in early childhood. Psychomotor regression was not reported. Behavioral disturbances occurred in two participants (#1 and #2), including attention deficit hyperactivity disorder (ADHD), irritability, immature social skills, and anxiety. Mild sleep disturbances were reported in patient #2, whereas an isolated epileptic episode occurred in participant #3 at the age of 1 year. Brain magnetic resonance imaging (MRI) was normal in two of the three tested participants (#1 and #3), while nonspecific findings were observed in individual #4 (Table 1).

Table 1.

Genetic and clinical features of CACHD1 participants.

#1 (family I) #2 (family II) #3 (family III) #4 (family III) #5 (family IV) #6 (family IV)
Age, gender 13.5 y, F 8.6 y, M 8 y, M 2 y, F 31+5 we, M 22+2 we, M
CACHD1 variant(s) (NM_020925.4) [c.1783–1G>A; c.2387+1G>A] [c.261+2T>C; c.648del (p.Ile217Serfs*13)] c.274dup (p.Ile92Asnfs*52) c.274dup (p.Ile92Asnfs*52) [c.277C>T (p.Arg93*); c.460C>T (p.Arg154*)] [c.277C>T (p.Arg93*); c.460C>T (p.Arg154*)]
Inheritance Comp het Comp het Hom Hom Comp het Comp het
Allele frequency (gnomAD, v3.1.2) 0; 0 0.00000657; 0 0 0 0.00000401; 0.0000402 0.00000401; 0.0000402
ACMG/AMP class (criteria) Pathogenic (PVS1, PM2, PP3); Pathogenic (PVS1, PM2, PP3) Pathogenic (PVS1, PM2, PP3); Pathogenic (PVS1, PM2, PP3) Pathogenic (PVS1, PM2, PP3) Pathogenic (PVS1, PM2, PP3) Pathogenic (PVS1, PM2, PP3); VUS - Likely pathogenic (PVS1, PP3) Pathogenic (PVS1, PM2, PP3); VUS - Likely pathogenic (PVS1, PP3)
Psychomotor delay (HP:0001263) No No Yes Yes NA NA
Cognitive impairment (HP:0100543) Yes, mild Yes, mild Yes, mild No NA NA
Seizures (HP:0001250) No No No Yes NA NA
Neuropsychiatric features (HP:0000708) Anxiety; irritability; ADHD; poor social skills Anxiety; sleep disorder No No NA NA
Facial dysmorphism (HP:0001999) Yes Yes Yes Yes Yes Yes
Ear abnormalities (HP:0000598) Microtia, posterior rotation, preauricular tags, helix hypoplasia, uplifted earlobes Overfolding of superior helices, preauricular skin tags No Preauricular skin tags Displacement, dysplastic outer ear, preauricular skin tags Displacement, dysplastic outer ear, preauricular skin tags
Hearing loss (HP:0000365) No No No No NA NA
Eye abnormalities (HP:0000478) Nasolacrimal duct obstruction; strabismus; epibulbar lipodermoid Strabismus, blepharitis Peters anomaly, epibulbar dermoid Coloboma NA NA
CMDTs (HP:0025031) ARM with anal displacement rectoperineal fistula ARM with anal atresia and recto-urethral fistula No ARM Esophageal atresia (Vogt Type 2) No
Genital abnormalities (HP:0000078) No Hypospadias Cryptorchidism, inguinal hernia Clitoromegaly No No
Renal defects (HP:0000077) No Unilateral renal agenesis No No Hydronephrosis due to urethro-renal obstruction Hydronephrosis due to urethro-renal obstruction
Cardiac abnormalities (HP:0001627) No PDA and PFO No Pulmonary stenosis, VSD No No
Abnormal ECG (HP:0003115) No No No No No No
Musculoskeletal features (HP:0033127) Scoliosis, short neck, broad halluces, flat feet Bilateral Perthes disease, proximal thumbs insertion No Left forefoot adduction No No
Other clinical features Trigonocephaly (HP:0000243); obesity (HP:0001513); hemifacial microsomia (HP:0011332) No No Relative enlargement of neurocranium (HP:0002683) Hypertrichosis (HP:0000998), accessory spleen (HP:0001747) Hypertrichosis (HP:0000998), accessory spleen (HP:0001747)
Brain MRI Normal NA Normal Nonspecific findings NA NA
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ACMG/AMP, American College of Medical Genetics and Genomics/Association for Molecular Pathology; ADHD, attention deficit-hyperactivity disorder; ARM, anorectal malformation; CMDT, congenital malformations of the digestive tract; Comp het, compound heterozygous; ECG, electrocardiogram; Hom, homozygous; NA, not applicable; PDA, patent ductus arteriosus; PFO, patent foramen ovale; PM, pathogenic moderate; PP, pathogenic supporting; PVS, pathogenic very strong; VSD, ventricular septal defect; we, weeks; y, years.

Figure 1. Genetic and clinical aspects of affected individuals harboring CACHD1 variants.

Figure 1.

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(A) Location of CACHD1 variants in relation to exonic location (top); and domain structure (bottom; GenBank: NM_020925.4, NP_065976.3). CACHD1 consists of an exofacial N-terminus, a von Willebrand factor A (VWA) domain, two bacterial chemosensory-like cache domains, a short hydrophobic transmembrane domain, and an intracellular C-terminus. Exonic variants affect exons 3, 4, and 6. Intronic variants localize in introns 2, 12, and 16. Most variants affect the early portion of the gene, predicted to lead to premature transcription termination and putative nonsense-mediated mRNA decay. Numbers under the protein schematic indicate amino acid numbers. Abbreviations are as follows: Cache, Ca2+ channel and chemotaxis receptor; CR, cysteine rich; HR, histidine rich; MIDAS, metal ion-dependent adhesion site; NLS, nuclear localization signal; TM, transmembrane domain. Locations of domains are approximate based on data from www.Uniprot.org. (B) Pedigrees of the reported families with the segregation patterns of CACHD1 variants. Open shapes, unaffected individuals; filled shapes, affected individuals; square, male; circle, female, triangle, pregnancy not carried to term; wt, wild type. (C) Clinical photographs. Individual #1 at 13 years shows sparse hair, medially sparse eyebrows, a pit on the left cheek, and small and posteriorly rotated ears with preauricular tags, underdeveloped crus of the helix, and uplifted earlobes. Individual #2 at 1 year shows bilateral preauricular skin tags associated with overfolding of the superior helices. Individuals #5 (31+5 weeks) and #6 (21+5 weeks) show long and thick eyebrows, periorbital rings, palpebral edema, low-set ears with dysplastic outer ear and bilateral preauricular skin tags, and macroglossia. (D) Graph summarizing the distribution of the most common clinical features (present in at least two cases) in the reported cohort. Abbreviations: CMDTs, congenital malformations of the digestive tract; DD, developmental delay; ID, intellectual disability; NA, not applicable.

All cases displayed dysmorphic facial features that included hypo- or hypertrichosis, medially sparse eyebrows, and bulbous nose tip (Figure 1c). Minor dysmorphic features were reported in individuals #1 (widely spaced nipples) and #2 (nipple skin tag). Variable ear abnormalities were observed in five of six individuals, and consisted of microtia, ear displacement or malrotation, hypo-dysplasia of the outer ear, preauricular skin tags, and uplifted earlobes. Four participants (#1–4) showed variable congenital eye anomalies, ranging from developmental defects (e.g., obstruction of the nasolacrimal ducts and bilateral glaucoma with buphthalmos) to benign tumors of choristomatous nature, such as epibulbar dermoid (individual #3) and lipodermoid (individual #1).

Congenital anomalies in different organ systems were common (Table 1; Supplemental Table 1). Five participants showed genitourinary abnormalities, of which three had renal involvement (unilateral renal agenesis in individual #2; uretero-renal obstruction leading to hydronephrosis in individuals #5 and #6); three cases presented with genital malformations, such as hypospadias (individual #2), cryptorchidism associated with inguinal hernia (individual #3), and abnormal enlargement of the clitoris associated with a distal skin tag (individual #4). Congenital malformations of the digestive tract (CMDTs) were diagnosed in three cases. Two participants (individuals #1 and #2) showed a similar pattern of anorectal malformations (ARM) consisting of anal displacement or atresia combined with rectoperineal fistula, whereas esophageal atresia was observed in participant #5. Musculoskeletal features were less common and included scoliosis (individual #1), foot malformation (individuals #1 and #4), and Perthes disease (individual #2). Cardiac involvement consisted of patent ductus arteriosus (PDA) and patent foramen ovale (PFO) in individual #2, and peripheral pulmonary artery stenosis (PAS) and ventricular septal defect (VSD) in individual #4. No rhythm abnormalities were detected.

Identification and in silico analysis of CACHD1 variants

We investigated each affected individual for copy number variants (CNVs) and single nucleotide variants (SNVs). CMA was uninformative for individuals #1–5; individual #5 had a normal karyotype, and individual #6 did not undergo chromosomal assessment (Supplemental Table 1). However, ES led to the identification of biallelic putative loss-of-function variants in CACHD1 (GenBank ID: NM_020925.4) segregating with disease in all pedigrees: c.1783–1G>A and c.2387+1G>A in family I; c.261+2T>C and c.648delC; p.(Ile217Serfs*13) in family II; homozygous c.274dup; p.(Ile92Asnfs*52) in family III; and c.277C>T; p.(Arg93*) and c.460C>T; p.(Arg154*) in family IV (Figure 1a–b; Table 1; Supplemental Table 1).

All variants are rare (allele frequency ranging from 0 to 0.0000402), are absent in the homozygous state in gnomAD, and are predicted to be pathogenic by multiple in silico tools (Table 1; Supplemental Table 2). The variants are dispersed across the CACHD1 locus, though four of the six changes (two frameshift and two stop gain variants) cluster in the region from intron 2 to exon 6. All variants are predicted to result in the loss of protein function either through nonsense-mediated mRNA decay (NMD) or the formation of an unstable truncated transcript. According to gnomAD, the probability of being loss-of-function intolerant (pLi) of CACHD1 is 0. However, the Loss-of-function Observed/Expected Upper-bound Fraction (LOEUF) score is 0.31 and the Z score for pLoF variants is 4.279, leading to a 100% probability of intolerance to recessive loss-of-function variants (https://varsome.com/gene/hg38/cachd1). Furthermore, the screening of our in-house database of 6,000 control exomes did not identify loss-of-function genotypes comparable to those observed in the reported individuals (Supplemental Table 3). Thus, our sequencing data indicates that mutations in CACHD1 result in the clinical symptoms observed in this patient cohort.

CACHD1 depletion disrupts neurogenesis in human cells

It is unclear how CACHD1 ablation from early neural cells could affect the mechanisms underlying brain development. Fetal neurogenesis relies on normal NPC proliferation and differentiation dynamics. To test the role of CACHD1 in neurogenesis, we depleted CACHD1 expression in H1 human embryonic stem cell (hESC)-derived NPCs that were generated using the SNaP protocol8 (Figure 2a). Two CACHD1-targeting sgRNAs were delivered separately to assess the reproducibility of effects, while one non-targeting (NT) sgRNA was used as a control. TIDE analysis revealed high mosaicism of over 95% editing efficiency for both guide RNAs (Supplemental Figure 1a–b).

Figure 2. CACHD1 depletion alters NPC proliferation and differentiation.

Figure 2.

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(A) Schematic describing the workflow of NPC cellular. (B) Quantification of the percentage of HuCD+ post-mitotic neurons in Day 30 differentiation cultures from control and CACHD1-edited SNaP lines. Representative images depict nuclei in blue and HuCD in green. Scale bar = 50 μm. (C) Quantification of the percentage of SOX2+ NPCs in Day 30 differentiation cultures from control and CACHD1-edited SNaP lines. Representative images depict nuclei in blue and SOX2 in green. Scale bar = 50 μm. (D) Schematic describing the production of three-dimensional (3D) neurospheres derived from two-dimensional SNaP cultures. (E) Lightsheet imaging of a Day 7 SNaP-neurosphere stained with phospho-Vimentin (NPC marker) and MAP2 (neurite marker). (F) Representative bright field images of control and CACHD1-edited neurospheres at day 15 post-plating. Scale bar = 1 mm. (G) Quantification of Panel F. Data are represented as mean ± S.D.

We allowed NPCs to spontaneously differentiate for 30 days to measure differentiation potential. CACHD1-edited cell lines showed reduced percentages of HuCD+ post-mitotic neurons, suggesting deficits in neuronal differentiation (Figure 2b). Measurements of NPC content revealed a modest but significant increase in SOX2+ progenitors in CACHD1 sgRNA#2 conditions relative to controls, while sgRNA#1 targeted cells showed a non-significant trend towards an increase of NPCs (Figure 2c). These findings corroborate recent analysis of CACHD1-depleted human cerebral organoids8 and further suggest that CACHD1-disrupted cells have an impaired ability to transition from proliferative NPCs to post-mitotic neurons.

Genome-wide CRISPR-Cas9 screening in 2D cultures previously identified CACHD1 as a regulator of NPC proliferation8. To test the impact of CACHD1 depletion under conditions of relevant cytoarchitecture and cell-type diversity16, we developed a protocol for generating 3D neurospheres from SNaPs (Figure 2d). Lightsheet imaging of day 7 unedited neurospheres immunostained with the NPC marker phospho-Vimentin (phVim) and the neurite marker MAP2 showed that both progenitors and early neuronal cells were present (Figure 2e). We further characterized the cellular composition of SNaP-derived neurospheres through scRNA-seq. Seven days post-neurosphere formation, we found 12.5% of the cells expressed transcripts enriched in post-mitotic neurons such as ELAVL4 and STMN2 (Group 4) while the remaining were either MKI67+ mitotic cells (Group 1, 34.3%) or progenitor cells that expressed PAX6 and SOX2 (Group 2, 28.2%; Supplemental Figure 2a–e). A subset of the quiescent state progenitors (Group 3, 25.0%) expressed genes that are typically upregulated under hypoxic conditions, such as BNIP3 and SLC2A1. Together, these findings support the utility of our SNaP-derived neurospheres for 3D modeling of NPC proliferation.

We produced neurospheres from CACHD1-depleted and NT sgRNA control SNaPs and found CACHD1 sgRNA #1 and #2 neurospheres were significantly larger than controls (Figure 2f–g) indicative of a hyperproliferative NPC phenotype. Collectively, our results indicate that CACHD1 plays a critical role in NPC cellular functions that occur during the earliest stages of brain development, and its genetic ablation could contribute to profound neurodevelopmental disorders.

Loss of CACHD1 alters neurodevelopmental gene expression programs

To understand the molecular mechanisms affected by CACHD1 loss, we harvested NPCs for bulk RNA sequencing analysis. We detected 432 significant differentially expressed genes (DEGs) between the control and CACHD1 sgRNA#1, and 1,924 DEGs between the control and sgRNA#2 (Figure 3a). Overall, 382 dysregulated genes (180 upregulated and 202 downregulated) were shared between the two datasets (Figure 3b–c). Enrichment analysis of gene ontology (GO) terms was conducted on these DEG lists to identify dysfunctional biological functions.

Figure 3. Genetic ablation of CACHD1 impacts expression of key neurodevelopmental genes and signaling pathways.

Figure 3.

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(A) Venn diagram depicting the overlap of differentially expressed genes (DEGs) between the two CACHD1-depleted NPC lines. (B-C) Volcano plots of DEGs, comparing each CACHD1-depleted line to NT control. Open circles reflect DEGs consistent in both CACHD1-depleted lines relative to NT control. Positive mean log2 fold change (FC) refers to genes that are upregulated in CACHD1-edited cells. Statistical significance is defined as Benjamini-Hochberg adjusted p-values <0.05. Vertical dashed lines represent log2FC = 0.6 (FC ~ 1.5 fold), while horizontal dashed lines represent the adjusted p-value threshold of p <0.05. DEGs that pass both the significance and FC thresholds are colored in red (upregulated) or blue (downregulated). Genes in green are known Wnt pathway genes. (D-E) Gene ontology (GO) term analysis of 382 DEGs; (D) downregulated and (E) upregulated genes in CACHD1-depleted lines.

Genes involved in broad neurobiological processes such as nervous system development (GO:0007399), neurogenesis (GO:0022008), synapse structure and activity (GO:0050803) were dysregulated in depleted cells (Figure 3d–e; Supplemental Data File 1). Key neurodevelopmental genes showed dramatic changes in expression, including SIX3 (log2FC = −4.62), HES1 (−1.74), CRABP2 (1.83), WNT5B (1.99), and NEUROD4 (2.51). Interestingly, our list of 382 DEGs showed significant overlap with high confidence autism (p=7.30e-04, odds ratio=2.1) and developmental delay risk genes (p=7.20e-04, odds ratio=3.5) such as CNTNAP2, GRIN2B, and PTEN, indicating potential biological convergence with established neurodevelopmental disorder risk factors.

Next, we set out to better understand the molecular contributors to the defective neurogenesis phenotypes we observed. Downregulated genes were enriched for such GO terms as regulation of neurogenesis (GO:0050767), neuron differentiation (GO:0045664), and negative regulation of cell population proliferation (GO:0008285), while upregulated genes were involved in neuroblast proliferation (GO:0007405), neuron differentiation, and cell division (GO:0051302). The downregulation of negative regulators of growth, including PTEN (log2FC=−0.45), MAPK21 (−0.67), and CDKN1C (−0.77) could explain the hyperproliferative phenotype. CDKN1C downregulation is also of interest given this gene’s role in promoting terminal differentiation of NPCs17. The reduced expression of differentiation-stimulating genes like CDKN1C, SOX4, and SOX11 could explain the lower differentiation potential of CACHD1-depleted cells.

Our data also highlight a potential role for CACHD1 in the Wnt signaling pathway, which regulates the balance between NPC proliferation and differentiation18. Downregulated genes were enriched for negative regulators of Wnt (GO:0030178), while upregulated genes were enriched for positive regulators (GO:0090263). These reciprocal effects suggest that the Wnt pathway may be overactivated in CACHD1-depleted cells, which is known to enhance proliferation and impair differentiation of NPCs19.

Beyond neurodevelopment, we detected abnormalities in other processes that may be relevant to our patient cohort. For example, our DEGs were enriched for genes involved in development of the head (GO:0060322), heart (GO:0007507), kidney (GO:0001822), skeletal system (GO:0001501), and urogenital system (GO:0001655; Figure 3e). Our analysis also revealed defects in molecular processes involved in pancreatic function (GO:0003310) and formation of amyloid-beta plaques (GO:1902003), the latter of which plays a central role in Alzheimer’s disease pathology. Collectively, these results suggest that disruption of CACHD1 expression impacts essential developmental processes and nominate gene expression programs that may underlie CACHD1-associated defects in neuronal and non-neuronal tissues.

Homozygous cachd1 mutant zebrafish larvae recapitulate human craniofacial dysmorphism

In vitro human models are a powerful tool for studying the mechanisms underlying disease, but are not able to recapitulate complex systems and structures, such as craniofacial development. To further establish the physiological relevance of CACHD1 dysfunction to human disease, we generated a zebrafish model. Reciprocal BLAST with human CACHD1 protein identified a single cachd1 ortholog in zebrafish (87% identity, 94% similarity; Figure 4a). cachd1 has robust mRNA levels detectable from the zygote stage, which persist in whole larval RNAseq data until at least 5 dpf20,21. We targeted exon 9 of the cachd1 locus by injecting sgRNA and Cas9 protein into the cell of 1-cell stage embryos. Further molecular characterization of F0 mosaic mutants showed moderate mosaicism (47% mosaicism with insertion/deletion events proximal to the PAM site in F0s, n=5 embryos per condition, 24 clones per embryo). F0 animals were outcrossed with WT (+/+) mates, and we identified an F0 founder carrying a 16 bp indel (p.(Phe452Leufs*3); Figure 4b). We in-crossed F1 heterozygous (+/−) mutants to generate an F2 generation for subsequent molecular validation. To confirm efficient ablation of cachd1 transcript in homozygous mutants (−/−), we performed qPCR on total RNA obtained from genotype-matched 2 dpf larvae (genotypes: cachd1+/+, cachd1+/−, cachd1−/−); we observed a significant reduction (~70–80%) in cachd1 RNA levels in homozygotes compared to WT siblings (p< 0.0001 versus WT; Figure 4c).

Figure 4. Ablation of zebrafish cachd1 by CRISPR-Cas9 results in craniofacial abnormalities.

Figure 4.

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(A) Schematic of zebrafish cachd51 transcript (GRCz11, Ensembl transcript ID: ENSDART00000087964.7) generated by Exon-Intron Graphic Maker (http://wormweb.org/exonintron). The sgRNA target site on exon 9 is indicated by a red triangle. Scale bar, 10 kb. (B) Representative sequence chromatograms of cachd1 +/+ (wild type), cachd1+/− (heterozygous mutant), cachd1−/− (homozygous mutant) are shown. Protospacer adjacent motif (PAM) is indicated by a red box for each chromatogram. Mutants harbor a 16 bp deletion (19 bp deletion and 3 bp insertion) that results in a frameshift and putative protein truncation (p.Phe452LeufsTer3). (C) Bar graph showing relative mRNA expression of cachd1 in genotype-matched 2 day post-fertilization (dpf) larvae generated from F1 in-crosses. n=20 per batch, three technical replicates per experiment, eight biological replicates (One way ANOVA; Tukey’s multiple comparisons test). Tails were used for genotyping while RNA was extracted from the matched heads for quantitative PCR analysis. Relative expression was normalized to gapdh and statistical differences were calculated using One way ANOVA [F (5, 42) = 50.39; P value<0.0001]; Tukey’s multiple comparisons test’s p-values for mutants versus +/+ are indicated above each bar. Error bars represent standard error of the mean. (D) Schematic of 3 dpf zebrafish larva showing a ventral view of craniofacial cartilage structures. Abbreviations: Meckel’s cartilage (mk, blue), palatoquadrate (pq, yellow), ceratohyal (ch, orange), hyosymplectic (hs, gray), ceratobranchial (cb, green). (E) Representative images of −1.4col1a1:egfp;cachd1 larvae imaged live at 3 dpf. Top: Bright field lateral images of wild type and homozygous mutants. Scale bar, 200 μm. Bottom: Fluorescent ventral images of wild type and homozygous mutants. Region of interest (ROI) area, as outlined in red and bordered by ch, pq and mk, was measured to detect statistical differences. Scale bar, 100 μm. (F) Quantification of ROI indicated in panel (E); AU, arbitrary units. Statistical differences were calculated using an unpaired t-test (n=50–70 larvae per condition, repeated). Biological replicates (1 and 2 as indicated in x-axis labels) were obtained from different parental pairs with the investigator was masked to the experimental conditions. Regardless of their parental genotype, all embryos obtained were morphologically similar and no animal was excluded from imaging and quantification. Error bars represent standard deviation of the mean.

Facial dysmorphisms are morphological features in humans bearing biallelic CACHD1 variants (Table 1; Supplemental Table 1; Figure 1c), prompting us to evaluate orthologous structures in zebrafish mutant larvae according to our in vivo imaging paradigm22,23(Figure 4d). Using F2 adult siblings, we in-crossed either WT or homozygous mutant animals harboring the −1.4col1a1:egfp transgenic cartilage reporter and simultaneously performed live lateral bright field imaging and ventral imaging of fluorescent signal in F3 larvae at 3 dpf. We observed no gross morphological abnormalities (Figure 4e, top). However, quantification of the mandibular area encompassed by ceratohyal, palatoquadrate and Meckel’s cartilage showed a significant reduction in the ROI area in cachd1−/− compared to cachd1+/+ larvae (p<0.0001; Figure 4e–f). These data suggest that dysfunction of cachd1 leads to cartilage patterning defects.

DISCUSSION

Defects in calcium channels have been implicated in several syndromic neurodevelopmental conditions24. VGCCs play crucial roles during neurodevelopment, including neural cell survival, neurite extension, and radial migration24. Pathogenic variants in genes encoding the α1 subunits of VGCCs result in heterogeneous neurodevelopmental features with or without other syndromic conditions25. Here, we identified six individuals harboring seven putative loss-of-function variants in CACHD1—which encodes a protein that interacts directly with VGCCs—presenting with a rare neurodevelopmental syndromic condition characterized by developmental delay, learning disabilities, facial dysmorphism, and extra-neurological manifestations featuring oculo-auricular abnormalities, genitourinary defects, and congenital malformations. In vitro human neural models of CACHD1 depletion displayed dysregulated Wnt signaling, which is in alignment with a recent report that nominates CACHD1 as a Wnt regulator in the developing brain26; it remains to be determined if Wnt dysfunction directly contributes to the altered NPC proliferation and differentiation we observed. Zebrafish cachd1 mutants displayed cartilage patterning defects, which is a proxy for human facial dysmorphic features. Collectively, our in vitro and in vivo findings reflect the developmental defects observed in human patients, nominating CACHD1 as a contributor to human brain and craniofacial development.

The CACHD1-associated cellular defects we observed align with the established role of VGCCs in neurogenesis29,30, and the clinical manifestations of CACHD1 variants—such as psychomotor development delay (2 of 4 cases), cognitive disability (3 of 4 cases), and neuropsychiatric features (2 of 4 cases)—are shared with calcium channelopathies and hint at potential converging disease biology31,32. For example, Timothy Syndrome is caused by pathogenic variants in CACNA1C, which encodes the α1C subunit of Cav1.2. This rare condition is characterized by neurodevelopmental delay, autism, and epilepsy, as well as syndactyly, prolonged QT interval, variable congenital heart defects, and facial dysmorphisms27,32. Similar neurodevelopmental and psychiatric features have also been associated with CACNA1G (Cav3.1) variants, as have variable craniofacial anomalies and skeletal defects28,33,34. Abnormal CACHD1-mediated regulation of these channels could potentially contribute to the neuropsychiatric symptoms observed in our patients.

Historically, investigations of VGCCs and their effectors have focused on their physiological relevance to excitable cells that can fire action potentials; their contribution to the development and function of non-excitable cells has received limited attention35,36. CACNA1C and CACNA1G variants play causal roles in craniofacial defects, exemplifying a developmental role for VGCCs in non-excitable tissues27,28,32. Deleterious variants in ion channel genes contribute to the morphological development and function of non-excitable cells in various animal models, and can result in congenital structural abnormalities37. For example, altered Cav1.2 channel activity leads to defects in jaw development in mice and zebrafish38. Accordingly, our cachd1 zebrafish mutants displayed cartilage patterning defects, and our RNA-seq analysis of in vitro human NPCs found dysregulation of transcripts involved in head and skeletal system development. Thus, the CACHD1 human and animal models we describe could serve as important systems for further elucidation of the roles of VGCCs in non-excitable cell morphogenesis. Future studies are required to elucidate the impact of CACHD1 dysfunction on critical craniofacial processes, such as neural crest cell formation and migration.

Some of the extra-neurological features observed in our patients overlap with oculo-auriculo-vertebral spectrum (OAVS; also known as Goldenhar syndrome), a rare condition characterized by malformations of the ears, eyes, and spine (Supplemental Figure 3). CNVs in different genetic loci have been identified in some patients, but the etiology of OAVS remains elusive40. CACHD1 has not been linked to this disorder, but it should be noted that OAVS minimal diagnostic criteria include features observed in our cohort such as microtia (small external ear), facial asymmetry due to hemifacial microsomia (i.e., one half of the face does not develop fully), and epibulbar dermoids/lipodermoids (benign growths in the eye). In mice, the loss of Cachd1 disturbs Ca2+ homeostasis in the endolymph of the inner ear, leading to secondary membranous labyrinth dilation and audio-vestibular dysfunction41. Although hearing loss and balance impairment were not observed in our patients, it is possible that mild defects could be detected through specific tests, such as video head impulse test, Computerized Dynamic Visual Activity, high-resolution computed tomography, and MRI. Subsequent studies could investigate the potential involvement of CACHD1 variants in the genetic susceptibility of OAVS and inner ear dysfunction.

CACHD1 may regulate key aspects of neurodevelopment independent of the modulation of VGCC activity42, as CACHD1-like α2δ isoforms are known to regulate synaptic function, GABAA receptor abundance, and axonal wiring in a non-VGCC-dependent manner7,43,44. Furthermore, protein products formed after CACHD1 cleavage by γ-secretase and the beta-site APP cleaving enzyme 1 (BACE1)—which is an essential catalyzer of the first step of pathogenic amyloid beta (Aβ) peptide generation in Alzheimer’s disease— modulate signal transduction and gene expression45.

CACHD1 has also been suggested in the pathogenesis of complex human conditions not observed in our patient cohort. Specifically, CACHD1 may contribute to the susceptibility to diabetes mellitus (DM) type 1 and has been implicated in the hepatocarcinogenesis associated with DM and Non-Alcoholic Steatohepatitis (NASH)46,47. It is tempting to speculate that an abnormal transcriptional regulatory function may contribute to at least some of the extra-neurological developmental defects observed in our patients.

We employed ES to investigate the etiology of neurodevelopmental symptoms observed in our cohort and identified biallelic variants in CACHD1 as the potential cause. While some LoF CACHD1 variants may be observed in healthy controls in gnomAD, these changes never occur in trans in the same individual. CACHD1 has a low LOEUF score (0.31) and high Z score for pLoF variants (4.279), suggesting intolerance to loss of function. Together with the results obtained from our functional studies, these data support the biallelic loss of CACHD1 as the cause of the novel neurodevelopmental syndrome observed in our patients. We acknowledge that the co-occurrence of variants with milder functional impact in modifier genes or non-coding regions may influence the phenotype expressivity of monogenic neurodevelopmental disorders, and that thorough genomic analysis may be helpful to detect these co-occurring variants. Though we were unable to investigate the whole genomes of our study’s participants, future broader genomic approaches could further dissect the genetic aspects of CACHD1-related disease.

In summary, our data expand the spectrum of human disorders related to VGCC function and suggest that CACHD1 participates in the refinement of cognitive function and morphogenetic processes in several organ systems.

Supplementary Material

Supplemental Table 2
Supplemental Table 1
Supplemental Data File 1
Supplemental Table 3
Supplemental information

ACKNOWLEDGEMENTS

We are grateful to the patients and families who participated in this investigation. We sincerely thank Jill A. Rosenfeld for the valuable cooperation and support. We also acknowledge the Telethon Undiagnosed Disease Program (TUDP) for technical and scientific support.

FUNDING STATEMENT

M.F.W. is supported by the NIMH/NIH (R00MH119327). E.E.D. is supported by funds from the NICHD/NIH (R01HD105868) and NIMH/NIH (R01MH106826). E.E.D. is the Ann Marie and Francis Klocke, MD Research Scholar.

Footnotes

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

ETHICS DECLARATION

This research study adheres to the principles in the Declaration of Helsinki and was approved by the medical ethical committee installed by Gaslini Children’s Hospital (Comitato Etico della Regione Liguria, approval code 163/2018). Written informed consent was obtained from parents or legal guardians of all the enrolled participants for clinical testing and publication of genetic and clinical data, as well as clinical photographs. Patient privacy was always prioritized and respected during the exchange of data among researchers and clinicians.

WEB RESOURCES

ACMG, https://www.acmg.net/ACMG/Medical-Genetics-Practice-Resources/Practice-Guidelines.aspx

ChopChop2, http://chopchop.cbu.uib.no/

ClinVar, https://www.ncbi.nlm.nih.gov/clinvar/

Combined Annotation Dependent Depletion (CADD), http://cadd.gs.washington.edu

DECIPHER: https://decipher.sanger.ac.uk

EMBL-EBI Expression Atlas, https://www.ebi.ac.uk/gxa/experiments/E-ERAD-475/Results

Ensembl, https://www.ensembl.org/index.html

Ensembl Variant Effect Predictor (VEP) pipeline, https://www.ensembl.org/info/docs/tools/vep/index.html

Exon-Intron Graphic Maker, http://wormweb.org/exonintron

FastQC, http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc

Gene Cards, http://www.genecards.org

Gene Matcher, http://www.genematcher.org

Genome Aggregation Database (GnomAD), http://gnomad.broadinstitute.org

Genomic Evolutionary Rate Profiling – GERP, http://mendel.stanford.edu/SidowLab/downloads/gerp/

Human Genome Variation Society, https://varnomen.hgvs.org

Leiden Open Variation Database (LOVD), https://www.lovd.nl

Mutation Assessor, http://mutationassessor.org/r3/

Mutation Taster, http://www.mutationtaster.org

Online Mendelian Inheritance in Man, http://www.ncbi.nlm.nih.gov/Omim

Polyphen-2, http://genetics.bwh.harvard.edu/pph2/

Proteomics DB, https://www.proteomicsdb.org

PubMed, http://www.ncbi.nlm.nih.gov/pubmed

RefSeq, https://www.ncbi.nlm.nih.gov/refseq

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

Splice AI, https://spliceailookup.broadinstitute.org

TIDE, https://tide.nki.nl/

UniProt, https://www.uniprot.org

UCSC Human Genome Database, http://www.genome.ucsc.edu

Varsome, https://varsome.com

DATA AVAILABILITY

The authors declare that all data generated or analyzed during this study and supporting the findings of the study are available within the paper and its supplementary information files. Genomic data included in the study are available in the Leiden Open Variation Database (LOVD) at https://databases.lovd.nl/shared/variants/CACHD1.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Table 2
NIHMS2059687-supplement-Supplemental_Table_2.xlsx (11.5KB, xlsx)
Supplemental Table 1
NIHMS2059687-supplement-Supplemental_Table_1.xlsx (14.6KB, xlsx)
Supplemental Data File 1
NIHMS2059687-supplement-Supplemental_Data_File_1.xlsx (105.9KB, xlsx)
Supplemental Table 3
NIHMS2059687-supplement-Supplemental_Table_3.xlsx (22.5KB, xlsx)
Supplemental information
NIHMS2059687-supplement-Supplemental_information.docx (1.8MB, docx)

Data Availability Statement

The authors declare that all data generated or analyzed during this study and supporting the findings of the study are available within the paper and its supplementary information files. Genomic data included in the study are available in the Leiden Open Variation Database (LOVD) at https://databases.lovd.nl/shared/variants/CACHD1.

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