Neural tube defects (NTDs) are severe malformations of the central nervous system that arise from failure of neural tube closure. HECTD1 is an E3 ubiquitin ligase required for cranial neural tube closure in mouse models. NTDs in the Hectd1 mutant mouse model are due to the failure of cranial mesenchyme morphogenesis during neural fold elevation. Our earlier research has linked increased secretion of extracellular heat shock protein 90 (eHSP90) to aberrant cranial mesenchyme morphogenesis in the Hectd1 model. Furthermore, overexpression of HECTD1 suppresses stress-induced eHSP90 secretion in cell lines. In this study, we report the identification of five rare HECTD1 missense sequence variants in NTD cases. The variants were found through targeted next-generation sequencing in a Chinese cohort of 352 NTD cases and 224 ethnically matched controls. We present data showing that HECTD1 is a highly conserved gene, extremely intolerant to loss-of-function mutations and missense changes. To evaluate the functional consequences of NTD-associated missense variants, functional assays in HEK293T cells were performed to examine protein expression and the ability of HECTD1 sequence variants to suppress eHSP90 secretion. One NTD-associated variant (A1084T) had significantly reduced expression in HEK293T cells. All five NTD-associated variants (p.M392V, p.T801I, p.I906V, p.A1084T, and p.P1835L) reduced regulation of eHSP90 secretion by HECTD1, while a putative benign variant (p.P2474L) did not. These findings are the first association of HECTD1 sequence variation with human disease and suggest that sequence variation in HECTD1 may play a role in the etiology of human NTDs.
Research Article
Identification and Functional Analysis of Rare HECTD1 Missense Variants in Human Neural Tube Defects
https://doi.org/10.21203/rs.3.rs-3794712/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 07 Mar, 2024
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Neural tube defects
HECTD1
extracellular HSP90
cranial mesenchyme
Neural Tube Defects (NTDs) are severe congenital malformations of the spine and brain. They are among the most common structural birth defects, with a global prevalence of 0.5 to 10 per 1000 live births (Greene and Copp 2014). The occurrence of NTDs in various populations is affected by genetics, geography, and the maternal diet (Li et al. 2006; Liu et al. 2016). For instance, in Northern China’s Shanxi Province in 2003, the frequency of NTDs was 138.7 per 10,000 births, 10-fold higher than in the United States and Europe (Li et al. 2006; Morris and Wald 2007; Williams et al. 2015). The diet in this region was deficient in folate, and the incidence of NTDs in this region was significantly reduced following a campaign to provide folic acid supplementation (Liu et al. 2016; Meng et al. 2015). NTDs arise in the first trimester of pregnancy from defects in neurulation, a process where the neural plate transforms into a tube to form the central nervous system (Wallingford et al. 2013). NTDs can occur at different axial levels, resulting in anencephaly in the head, spina bifida in the spine, or craniorachischisis with the complete failure of neural tube closure along the entire neural axis. Anencephaly and craniorachischisis are fatal, whereas spina bifida can result in significant disability.
While the causes of NTDs are polygenic and multifactorial, estimates suggest that ~ 70% of NTDs have a significant genetic component (Copp et al. 2015; Jorde et al. 1983; Lupo et al. 2017). For instance, the relative risk among first-degree relatives increases to 3% (Jorde et al. 1983), and NTDs are more frequent in some genetic syndromes and chromosomal anomalies (Copp et al. 2015; Lupo et al. 2017; Toriello and Higgins 1983). Genome-wide sequencing projects indicate that rare and novel variants in NTD candidate genes may contribute to NTDs in an oligogenic fashion (Chen et al. 2018c; Ishida et al. 2018). For example, screening a cohort of 90 cases with cranial NTDs from northeast England between 1992 and 2011 with a targeted exome sequencing panel of 191 genes identified 397 rare variants (Ishida et al. 2018). On average, NTD cases had nine rare/novel variants, three of which were predicted to be damaging. In contrast, case-control samples had an average of two novel/rare variants, with 1.5 predicted to be damaging (Ishida et al. 2018). In analyzing whole-genome sequencing (WGS) data from three different NTD cohorts (Han Chinese, Caucasian USA, and Middle Eastern/Qatar) with various NTD types, researchers found a higher occurrence of singleton loss-of-function (SLoFVs) variants among NTD cases than controls (Chen et al. 2018c). SLoFVs were defined as variants that appear only once in the 1000 genome project. Based on these findings, the authors suggest that the number of SLoFVs is a stable and reliable genomic indicator of NTD risk in humans, with nine SLoFVs a genomic threshold for NTD risk (Chen et al. 2018c).
Animal studies have identified hundreds of genes involved in forming the neural tube (Harris and Juriloff 2007, 2010; Ishida et al. 2018; Wilde et al. 2014). Searching for rare and novel sequence variants in these NTD candidate genes in NTD cases has been a powerful tool for revealing the genetic causes of human NTDs (Ishida et al. 2018; Wolujewicz and Ross 2019). Rare variants in genes under constraint are of particular interest in rare disease research due to the anticipated stronger effects (Gudmundsson et al. 2022). However, an analysis of the Genome Aggregation Database (gnomAD v4.0.0) (Chen et al. 2022; Karczewski et al. 2020) reveals that many NTD candidate genes tolerate missense variation (Supplemental Table 1). For instance, multiple VANGL1 missense variants have been identified in NTD cases and the pathogenesis of many of these variants was validated in experimental assays (Bartsch et al. 2012; Cai et al. 2014; Cheng et al. 2021; De Marco et al. 2014; Doudney et al. 2005; Fatima et al. 2022; Humphries et al. 2020; Iliescu et al. 2014; Iliescu et al. 2011; Kibar et al. 2009; Kibar et al. 2007; Merello et al. 2015; Reynolds et al. 2010; Tian et al. 2020a; Tian et al. 2020b; Wang et al. 2018). VANGL1 tolerates missense but not loss of function (LOF) variants with a missense Z score of 0.59 but a loss intolerance probability (pLI) score close to 1 (0.91) and a LOF observed/expected upper bound fraction (LOEUF) score of 0.53. This is consistent with the proposed digenic and multigenic origins of NTDs involving sequence variation in VANGL genes (Juriloff and Harris 2012; Torban et al. 2008; Wang et al. 2018; Zohn 2012; Zohn and Sarkar 2008). In contrast, only a handful of NTD candidate genes exhibit substantial selection against missense variation. Among these is HECTD1, a HECT domain E3 ubiquitin ligase that targets proteins for degradation or alters their function. Since causal LOF variants for Mendelian and severe complex diseases are enriched in 'mutation intolerant' genes (Agarwal et al. 2023), the strong selection against LOF and missense variants in HECTD1 provides support to the idea that deleterious sequence variation in the HECTD1 gene would significantly affect embryonic development and based on the mouse phenotype, disrupt neural tube closure (Sarkar and Zohn 2012; Zohn et al. 2007).
We originally identified the mouse Hectd1 gene in an ENU mutagenesis screen to identify genes required for neural tube closure (Kasarskis et al. 1998; Zohn et al. 2007). This novel ENU-induced Hectd1 mutant mouse model (openmind, opm) exhibited fully penetrant exencephaly in homozygous Hectd1opm/opm embryos and incomplete penetrance in heterozygotes (Zohn et al. 2007). Interestingly, depending on the mutation, 5–20% of heterozygous Hectd1 mutant mouse embryos showed exencephaly (Zohn et al. 2007). Our study of the developmental mechanism leading to exencephaly in the Hectd1 mouse model revealed that the defect arises from the abnormal morphogenesis of the cranial mesenchyme (Sarkar and Zohn 2012; Zohn et al. 2007), a process required to elevate the cranial neural folds (Morris-Wiman and Brinkley 1990a, b, c; Morriss and Solursh 1978a; Morriss and Solursh 1978b; Zohn and Sarkar 2012). Analysis of the pathways regulated by HECTD1 in the cranial mesenchyme implicated increased secretion of extracellular Hsp90 (eHsp90) as a likely cause of the cranial mesenchyme and neural tube closure defects in Hectd1 mutant embryos (Sarkar and Zohn 2012). Our research demonstrated that eHSP90 stimulates the migration of cranial mesenchyme cells, interfering with normal cranial mesenchyme morphogenesis and neural fold elevation. Likewise, the expression of HECTD1 in HEK293T cells suppresses the stressed-induced secretion of eHSP90 (Sarkar and Zohn 2012).
The present study identifies five case-specific missense variants in the HECTD1 gene from a Chinese NTD cohort. Based on our prior knowledge that NTDs in the Hectd1 mutant mouse model are due to elevated secretion of eHSP90 stimulating cranial mesenchyme migration (Sarkar and Zohn 2012), we utilize this as an assay to functionally test the impact of missense variants on HECTD1 function. Our findings indicate that all variants associated with NTDs showed activity loss, whereas a putative benign HECTD1 variant did not. These data suggest sequence variation in HECTD1 may contribute to human NTDs.
Human Subjects and Targeted Next-Generation Sequencing of HECTD1
The subjects of this study were previously described (Chen et al. 2018a; Chen et al. 2018b; Lei et al. 2019; Qiao et al. 2016; Shi et al. 2018; Ye et al. 2020). 352 NTD samples were collected from aborted fetuses and children with spina bifida from the 1990s to the 2010s in five Chinese provinces. Tissue samples were collected from 309 aborted fetuses (23.4 ± 6.7 weeks) with craniorachischisis, encephalocele, anencephaly, exencephaly, spina bifida, or more than one type of NTD. Whole blood samples were obtained from 43 children (6.4 ± 4.6 years) with spina bifida. 224 control samples (58.9% female, 40.6% male, 0.5% unknown) were ethnically and gender-matched unrelated healthy volunteers recruited from Shanxi (aborted healthy fetuses) and Shanghai (blood samples from healthy first-year college students) in China. Protocols were reviewed and approved by the Ethics Committee of the School of Life Sciences, Fudan University. The coding region of HECTD1 was sequenced by targeted next-generation sequencing as previously described (Qiao et al. 2016). Variants were assessed for predictive deleteriousness using SIFT (http://sift.jcvi.org/) and Mutation Taster2 (http://www.mutationtaster.org/). HECTD1 variants analyzed in this study were absent in controls, the 1000 genome project (http://www.1000genomes.org), or extremely rare in the Genome Aggregation Database (gnomAD; http://gnomad.broadinstitute.org/).
Cell Culture and DNA Transfection
HEK293T cells from American Type Culture Collection (CRL-11268) were maintained in high glucose DMEM media (Gibco 11995065) supplemented with 10% Fetal bovine serum and Penicillin-Streptomycin and grown in a 5% CO2 humidified incubator at 37°C. pCMV-HA-Hectd1 and pCMV-Hsp90a-Myc constructs were previously described (Sarkar and Zohn 2012). Aligent QuikChange II XL Site-Directed Mutagenesis (Aligent 200521) was used to introduce missense mutations confirmed by Sanger sequencing. CD63-pEGFP C2 (gift from Paul Luzio, Addgene plasmid 62964; https://www.addgene.org/62964/; RRID: Addgene_62964), pRK5-HA-GFP (HA-GFP was a gift from Carol Mercer (Addgene plasmid 137763; http://n2t.net/addgene:137763; RRID: Addgene_137763) (Stefely et al. 2020). Plasmids were prepared using the Qiagen Mini-Prep Kit (Qiagen 27104) and cut with PvuII-HF restriction enzyme (NEB R3151S) to confirm quality and concentration. HEK293T cells were transfected using Lipofectamine 3000 Reagent (L3000008) according to the manufacturer's instructions with either 730 ng pCMV-HA-Hectd1, 400 ng pCMV-Hsp90a-Myc, and 50 ng CD63-pEGFP C2 or 1 ug pCMV-HA-Hectd1 and 50 ng pRK5-HA-GFP.
Western Blot Analysis
Transfected cells were expanded to a 6-well plate 24 hours after transfection and lysed 48 hours post-transfection. Lysis was performed using IP Lysis Buffer (Pierce, 87787) with diluted 100x Halt Protease Inhibitor Cocktail (78429). Plates were rocked at 4°C for 10 minutes, scraped, and debris was separated by centrifuging 13,000 x g for 10 minutes at 4°C. The supernatant was collected, and concentration was quantified using a Pierce BCA Protein assay kit (23225) or Coomassie Protein Assay Reagent (Thermo Scientific 1856209). Lysate was prepared in NuPAGE 4x Sample Buffer (NP0007), NuPAGE Reducing Agent 10x (NP0004), heated at 70°C for 10 min, and resolved on 10-well NuPAGE 3 to 8%, Tris-acetate Mini Protein Gel (EA0375) with NuPAGE Tris-Acetate SDS Running Buffer (LA0041) to detect HA-HECTD1 or 10-well NuPAGE 4 to 12%, Bis-Tris Mini Protein Gel (NP0321) with the NuPAGE MES SDS Running Buffer (NP0002) to detect HA-GFP run at 150V for 1.5 hours. Protein was transferred using a low fluorescence PVDF Transfer Membrane (22860) at 20V for 1.5 hours. Transfer buffer was supplemented with NuPAGE Transfer Buffer 20x (NP0006), 10% Methanol, and NuPAGE Antioxidant (NP0005). Membranes were blocked with LiCOR Intercept Blocking Buffer (927–60,001) for 1 hour at room temperature. Primary antibodies were incubated overnight at 4°C at the following dilutions: HA-HECTD1 and HA-GFP were detected using anti-HA.11 (Clone 16B12, BioLegend) diluted 1:750 and 1:1000, respectively, in Intercept Antibody Dilutant (927–65,001). Secondary antibody IRDye 800CW donkey anti-mouse (92,632,212; 1:15,000 dilution) was incubated for 1 hour at room temperature. Blots were imaged using the LiCor Odyssey CLX imaging system and LI-COR software. Exported image files were processed to 800 channel only, inverted LUT, and integrated density was measured over 3 established Regions of Interest (ROIs). Relative expression was measured through averaged integrated density values and calculated by normalizing HA-HECTD1 expression to transfection control HA-GFP. Statistical analysis was performed using Prism GraphPad 9, and the significance was determined using the two-tailed Student’s t-test. Four replicates were used in the western analysis; p.A1084T is reported in triplicate due to a technical error. HA-HECTD1 and p.P2474L were assayed using the same method independently for three replicates.
eHsp90a Secretion Assay
Transfected cells were passaged 24 hours after initial transfection to a 6-well plate with a sterilized coverslip in the well. 24 hours after passaging cells, respective wells were treated with 10 micromolar N-acetyl- N-Acetyl-Leu-Leu-Nle-CHO (ALLN, BML-P120) for 1 hour. Cells were washed with 1x PBS with calcium and magnesium (Gibco 14040141) and fixed for 20 minutes with 4% Paraformaldehyde. Blocking buffer for primary antibodies and washes were formulated with 1x PBS (Sigma, P3813) and 1% heat-inactivated goat serum (16210064). Primary incubations were done for 1 hour at room temperature with Myc-Tag (71D10) Rabbit mAb (1:200 Cell Signal, 2278), and secondary incubations for 1 hour at room temperature in the dark with Hoechst 33342 Solution (1:500, 62249) and Alexa-Fluor Anti-Rabbit 555 (1:250, A-21428). Final washes were performed with a blocking buffer with 0.1% Triton - X100 to reduce excess signal. Coverslips were mounted using Fluoromount-G™ Mounting Medium (00-4958-02). Slides were imaged using the Leica TCS8 Confocal Microscope with a 63x-magnification oil immersion objective (NA = 1.4). Image acquisition was made blind by randomization of slides. Images were acquired on detection of CD63-GFP transfection control, and then parameters were adjusted for detection of extracellular Hsp90a or lack thereof. The maximum projection of the Z stack was applied to images in ImageJ. Images of cells acquired during blind acquisition had to exhibit standard nuclei shape and health to ensure intact plasma membrane. Final images were processed using a coding key and scored by five independent reviewers to reach a consensus on categorizing myc-eHSP90 staining as “No secretion,” “Moderate secretion,” or “Excessive secretion.” After the scoring was performed, slides were unblinded for statistical analysis. Chi-squared tests were performed to test significance. ALLN treatment conditions were assessed independently. Three replicates were performed, consisting of five frames per replicate for 15 frames per condition. Hectd1 and p.C2579G were n = 20 after including p.P2474L in a separate assay.
Multiple Sequence Alignment of HECTD1 Protein Sequences
To obtain the sequences for the graphical alignment and multiple sequence alignment of HECTD1, a series of blastp queries were performed strategically, moving through lower-order organisms. The subject sequence was obtained from UniProt as the canonical HECTD1 Homo sapiens protein sequence (Q9ULT8). Standard laboratory animal models were first queried (M. musculus, G. gallus, X. laevis, D. rerio, D. melanogaster, C. elegans). Additional queries were performed: opossums (taxid: 9265), bony fish (taxid: 7898), lampreys (taxid: 7745), cnidaria (taxid: 6073), sponges (taxid: 6040), and anasdipea (taxid: 6497). Named proteins and X1 isoforms (where applicable) were chosen as sequences for multiple sequence alignment. blastp (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE=Proteins) queries were performed using default parameters. Sequences were aligned using MUSCLE with the UPGMA clustering method, 1.20 hydrophobicity multiplier, -2.90 gap open, and Lambda of 24. Alignment was visualized utilizing SnapGene.
AlphaFold Rendering of Human NTD-Associated Variants
AlphaFold v2.3.0 (Jumper et al., 2021) renderings were performed on the Children’s National Research Institute High-Performance Computing cluster. The human canonical sequence (Uniprot Q9ULT8) was edited into multiple FASTA files with missense variants. Each missense variant FASTA protein sequence was run using the monomer_ptm preset and 9/8/2022 max template date. The zero-ranked PDB output for each variant was imported into YASARA and superimposed on the zero-ranked wild type PDB output. Residues of interest were transformed to the ball and stick view for structural and confidence score (b-factor/pLDDT) analysis. pLDDT scores are included for wild type and missense residues.
Targeted next-generation sequencing of HECTD1 in an NTD cohort identified five rare (minor allele frequency < 0.01) heterozygous missense variants (Table 1 and Fig. 1A). Evaluation of these variants across publicly available databases indicated that variants are either exceedingly rare or unique. The c. 1174A > G (p. M392V), c. 2716A > G (p. I906V), c. 2716A > G (p. A1084T) and c.5504C > T (p.P1835L) were rare, with gnomAD frequencies of 2.09e-6 (2.723e-6 in the European non-Finnish but not reported in the East Asian ancestry group), 1.86e-5 (2.233e-5 among the East Asian ancestry group), 7.97e-6 (1.389e-4 among the East Asian ancestry group), and 6.84e-7 (8.996e-7 in the European non-Finnish but not reported in the East Asian ancestry group), respectively. One variant, c.2402C > T (p.T801I), was novel and not found in the gnomAD dataset or among the ethnically matched control cohort. All but one variant (p. I906V) is predicted to be damaging/deleterious by PolyPhen-2 and SIFT variant effect prediction tools (Adzhubei et al. 2010; Ng and Henikoff 2003). As a control, this study also includes a reported (ClinVar) benign variant, p.P2474L (rs111683057). This variant has a gnomAD frequency of 1.1e-3, primarily in the African/African American ancestry group (2.043 e-2), with 9 homozygous individuals reported.
Table 1. HECTD1 variants tested in this study.
The HECTD1 missense variants alter conserved amino acids localized within defined and well-conserved functional domains of HECTD1 or amino acids just flanking these domains (Fig. 1A). Our previous results indicate that HECTD1 interacts with HSP90 through N-terminal armadillo and ankyrin repeats (Sarkar and Zohn 2012). SUN (Sad1-UNC-84 homology) domain and Basic tilted helix bundle (BTHB) domains can also mediate protein-protein interactions (Dilworth et al. 2017; Starr and Fridolfsson 2010). A multiple sequence alignment was performed (Fig. 1B) utilizing MUSCLE (MUltiple Sequence Comparison by Log- Expectation) to align the sequences of selected species (Starr and Fridolfsson 2010). All residues implicated in our missense variants were universally conserved among vertebrates, and several were also preserved in the invertebrate species surveyed.
AlphaFold analysis of HECTD1 protein structure suggests missense variants do not result in significant changes to tertiary or secondary structure (Fig. 2). The pLDDT scores do not indicate relevant deviations from the wild type structure. Differences in pLDDT scores are represented in coloration changes of the b-factor field in the 3D rendering of the PDB output and text output shown underneath each frame (Fig. 2). Significant conformational changes would have been indicative of destabilizing variants.
HECTD1 is a Highly Conserved and Constrained Gene
Sequence variation in human HECTD1 is under notable constraint. The gnomAD database indicates that loss of function variants are significantly underrepresented in the HECTD1 gene. HECTD1 loss of function variants have a LoF intolerant (pLI) score of 1 and a LOEUF score of 0.27 (50 observed vs. the expected 238.4 LOF variants). Since haploinsufficient genes often have a high pLI score and a low LOEUF score (Gudmundsson et al. 2022; Lek et al. 2016), these data indicate that HECTD1 could be a haploinsufficient disease gene. The HECTD1 gene is also highly constrained for missense variation with a Z score of 6.42 (1911 observed SNVs vs. 2849.1 expected SNVs). Moreover, HECTD1 has an RVIS (Residual Variation Intolerance Score) of -2.23, ranking among the 1.33% most intolerant to functional genomic changes of all human genes (Petrovski et al. 2013). These metrics suggest that missense variation and loss of function changes in HECTD1 are poorly tolerated compared to most human genes. Additionally, this high level of constraint could be consistent with mutations in HECTD1 causing disease in heterozygous individuals.
Table 2. Blastp results of HECTD1 against different species for comparative analyses in Figure 1B.
The p.A1084T Variant Showed Reduced Expression in HEK293T Cells
First, we tested if missense variants alter the expression levels of HECTD1 in HEK293T cells. Western blot analysis of transiently transfected HEK293T cells shows that some HECTD1 variants from human NTD cases reduced expression compared to the wild type HECTD1 (Fig. 3). The p.A1084T variant exhibited significantly reduced expression in three independent experiments (p < 1e-6), whereas the expression of the other NTD-associated variants and the benign p.P2474L variant was not significantly reduced.
HECTD1 NTD-Associated Variants Show Reduced Activity
Our previous studies demonstrate that increased secretion of extracellular HSP90 (eHSP90) through the exosome pathway in Hectd1 mutant cells stimulates abnormal Our previous studies demonstrate that increased secretion of extracellular HSP90 (eHSP90) through the exosome pathway in Hectd1 mutant cells stimulates abnormal migration of the cranial mesenchyme in an explant assay (Sarkar and Zohn 2012). We showed that overexpression of HECTD1 prevents eHSP90 secretion in HEK293T cells and in response to stress induced by treating cells with the protease inhibitor ALLN (Sarkar and Zohn 2012). Thus, this assay can functionally test whether NTD-associated HECTD1 missense variants retain the functionality of preventing eHSP90 secretion, a role related to NTDs. HEK293T cells were transfected with Myc-Hsp90, CD63-GFP, and the HA-Hectd1 variant constructs. CD63-GFP is a well-characterized maker of exosomes secreted from the cells and was included to identify transfected cells. Immunostaining was done without permeabilization with detergent to detect only extracellular Myc-HSP90. Multiple independent reviewers scored the assay for the presence and intensity of eHSP90 staining on the surface of CD63-expressing cells. As shown in representative images in Fig. 4A, data were scored as “No secretion,” “Moderate secretion,” or “Excessive secretion.” Variants p.M392V, p.I906V, and p.P1835L demonstrate activity loss indicated by increased frequency of cells scored with “moderate” or “excessive” secretion of eHSP90 (Fig. 4B, No ALLN). This experiment was repeated in the presence of the protease inhibitor ALLN, which stimulates the secretion of eHsp90 (Sarkar and Zohn 2012). Upon the addition of ALLN, all NTD-associated sequence variants, but not the benign p.P2474L variant, showed reduced activity as measured by an increased proportion of cells scored with “moderate” or “excessive” secretion of eHSP90 compared to the wild type HECTD1.
Table 3 Summary of functional analysis shown in Fig. 4 to determine the effect of human NTD-associated HECTD1 sequence variants on eHSP90 secretion. At baseline (No ALLN) or after treatment with ALLN when expressed in HEK293T cells
This study identified five case-specific rare missense variants in the HECTD1 gene in a well-described Chinese NTD cohort (Chen et al. 2018a; Chen et al. 2018b; Lei et al. 2019; Qiao et al. 2016; Shi et al. 2018; Ye et al. 2020). Our previous work demonstrated that HECTD1 encodes an E3 Ubiquitin ligase required for neural tube closure in mouse models (Zohn et al. 2007). We now show evidence that HECTD1 is a highly conserved gene across the animal kingdom. Moreover, the NTD-associated sequence variants identified in our study alter evolutionarily conserved amino acids within or adjacent to conserved functional domains of the protein. The missense variants identified in HECTD1 in NTD cases are extremely rare in reference population databases, further supporting the possibility of pathogenicity. To generate experimental evidence supporting pathogenicity, NTD-associated HECTD1 missense variants were expressed in HEK293T cells to determine if they altered protein expression or reduced gene function. Variant p.A1084T was expressed at significantly lower levels when transfected into HEK293T cells. All NTD-associated variants tested showed reduced activity in suppressing the secretion of eHSP90, a pathway regulated by HECTD1 previously linked to causing NTDs in our mouse model (Sarkar and Zohn 2012). Together, these data support a role for sequence variation in HECTD1 contributing to NTDs in humans.
We originally identified Hectd1 in a forward genetic screen in mice seeking new candidate genes for NTDs (Kasarskis et al. 1998; Zohn et al. 2005, 2007). Further study of our Hectd1 mutant mouse lines found that NTDs result not from defects in the neural tissue but from disruption of cranial mesenchyme morphogenesis required for cranial neural fold elevation (Sarkar and Zohn 2012; Zohn et al. 2007). Mutation of Hectd1 causes increased secretion of extracellular HSP90, resulting in altered morphogenesis of the cranial mesenchyme (Sarkar and Zohn 2012). The cranial mesenchyme lies underneath the cranial neural folds, and expansion of the cranial mesenchyme is believed to be required for the initial elevation of the cranial neural folds (Zohn and Sarkar 2012). Thus, in Hectd1 mutant mouse embryos, increased eHSP90 secretion results in abnormal movement of the cranial mesenchyme, disrupting neural fold elevation and leading to NTDs. In the present study, the ability of HECTD1 to suppress eHSP90 secretion was utilized as an assay to determine if NTD-associated sequence variants resulted in loss of HECTD1 activity. All five variants identified reduced the activity of HECTD1 to prevent eHSP90 secretion, demonstrating that the sequence changes reduce activity related to the etiology of NTDs.
The HECTD1 sequence variants identified in this study were found in the heterozygous state in NTD cases. While studies of our mutant mouse line causing NTDs utilized a homozygous model, mutation of one copy of Hectd1 can result in NTDs in our model (Sarkar and Zohn 2012; Zohn et al. 2007). Approximately 5% of heterozygous Hectd1opm mutant embryos exhibit exencephaly with a stop gain mutation at amino acid 144 (L144X) and 20% of heterozygous embryos with disruption of the HECT domain in the Hectd1Gt(XC266)Byg line show NTDs. Since mutation of the HECT domain of HECT E3 ligases creates a dominant-negative protein by preserving the ligase's interaction with substrates but preventing ubiquitination and degradation of substrates (Huibregtse et al. 1995; Treier et al. 1992), the Hectd1Gt(XC266)Byg mutation likely has some dominant-negative activity.
Analysis of the gnomAD population database further supports the idea that sequence variation in HECTD1 could cause disease in heterozygotes. We found that HECTD1 has a high pLI score and a low LOEUF score and is intolerant to missense variation consistent with a haploinsufficient disease gene (Gudmundsson et al. 2022; Lek et al. 2016). One limitation of our study is the lack of available trio sequence data to determine the inheritance pattern or if the sequence variants occur de novo. Notably, the ethnically matched control cohort analysis did not identify the variants, which are extremely rare in the Asian population.
Genetic analysis of NTDs indicates oligogenic inheritance with environmental influences (Caiaffa et al. 2023; Chen et al. 2018c; Copp and Greene 2010; Ishida et al. 2018). HECTD1 is a ubiquitin ligase that regulates many pathways required for neural tube closure by targeting proteins for degradation or altering their function. Thus, it is also possible that sequence variation in HECTD1 would interact with additional variants in these pathways to disrupt neural tube closure or contribute to human disease. HECTD1 plays a role in several signal transduction pathways essential for embryonic and fetal development, including Notch, Wnt, Retinoic acid, and estrogen receptor signaling (Chen and Greenwald 2014; Li et al. 2015; Oikonomaki et al. 2017; Sugrue et al. 2019; Tran et al. 2013). HECTD1 could broadly influence development by regulating the STRIPAK complex, chromatin remodeling, cholesterol export, and protein translation (Aleidi et al. 2015; Aleidi et al. 2018; Beard et al. 2016; Lampersberger et al. 2023; Li et al. 2015; Lu et al. 2022; Lv et al. 2021). HECTD1 influences proliferation, apoptosis, autophagy, DNA damage repair, and mitochondrial function (Beard et al. 2016; Bennett et al. 2020; Duhamel et al. 2018; Liao et al. 2023; Lu et al. 2022; Segref et al. 2014; Uemoto et al. 2022; Vaughan et al. 2022). HECTD1 is also involved in inflammation in astrocytes, microglia, and other cell types (Li et al. 2020; Liao et al. 2023; Tang et al. 2021; Zhang et al. 2020) and can regulate synaptic function by regulating the expression of glutamate transporter 1 (GLT-1) in astrocytes (Zeng et al. 2022). Intriguingly, the Chinese NTD cohort utilized in this study was collected in a region where folate deficiency is well documented (Liu et al. 2016; Meng et al. 2015). It would be intriguing to investigate whether the NTD-associated variants identified in our study reduce the activities of other HECTD1-regulated pathways. Additionally, we could explore if HECTD1 variants interact with genetic variation or folate deficiency to cause NTDs.
In conclusion, we detected five case-specific rare missense variants in the HECTD1 gene in a Chinese NTD cohort. All were functionally validated in assays demonstrating reduced protein expression of one variant and reduced function of all variants in an eHSP90 secretion assay linked to the mechanism of NTD formation in mouse models. Our results provide the first report of sequence variation in the HECTD1 gene potentially contributing to human disease and the etiology of human NTDs.
Acknowledgments. Genome Aggregation Database (gnomAD v4.0.0; https://gnomad.broadinstitute.org) and Online Mendelian Inheritance in Man, OMIM (https://omim.org/) were accessed on 12/22/23. Tatiana Ferebee assisted with database searched to assemble Supplementary Table 1.
Funding. This work was supported by R01HD098861 by NICHD (PI: I. Zohn) and utilized the molecular imaging core of the District of Columbia Intellectual and Developmental Disabilities Research Center (DC-IDDRC) Award P50HD105328 by NICHD (PI: V. Gallo).
Competing Interests. The authors have no relevant financial or non-financial interests to disclose.
Author Contributions. IEZ designed the study and wrote the paper, EO designed the study, performed the functional analysis of the variants and wrote the paper, and HY performed the sequencing and analysis. HL recruited the patients. All authors reviewed the paper and approved the manuscript.
Data Availability. The datasets generated during and/or analyzed in this study are available from the corresponding author upon reasonable request.
Ethics Approval. Protocols were reviewed and approved by the Ethics Committee of the School of Life Sciences, Fudan University.
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No competing interests reported.