Bi-allelic ATG4D variants are associated with a neurodevelopmental disorder characterized by speech and motor impairment

Patient enrollment and consent

Individual 1 (Family 1: II-2) was evaluated through the National Institutes of Health (NIH) Undiagnosed Diseases Program (UDP)38,39 and was enrolled in the protocol 76-HG-0238, approved by the National Human Genome Research Institute Institutional Review Board. His mother provided written informed consent. Individual 2 (Family 2: II-3) and her younger affected sister Individual 3 (Family 2: II-4) were clinically assessed and followed through the Provincial Medical Genetics Program at the British Columbia Women’s and Children’s Hospital (Vancouver, BC, Canada). Individual 2 (Family 2: II-3), her affected younger sister Individual 3 (Family 2: II-4), two unaffected siblings (Family 2: II-1 and Family 2: II-2), and mother (Family 2: I-2) were enrolled in the protocol 76-HG-0238, approved by the National Human Genome Research Institute Institutional Review Board for sample collection and molecular analyses. Their mother provided written informed consent for all individuals in this family. The authors affirm that human research participants provided informed consent for the publication of the images in Fig. 1.

Exome and/or genome sequencing analysis

Peripheral whole blood samples were collected, and DNA was extracted using CLIA-approved methods. Additionally, for Family 2, DNA samples were collected from several family members using the iSWAB Discovery Collection Kit (Mawi DNA Technologies, Hayward, CA) and the genomic DNA was extracted from the buccal swabs using the QIAamp DNA Mini Kit (Qiagen, Germantown, MD) for Sanger sequencing validation.

Initial trio clinical exome sequencing was performed on Individual 1 and his parents at GeneDx (Gaithersburg, MD) followed by quartet clinical genome sequencing at HudsonAlpha Genome Sequencing Center (Huntsville, AL) through the Undiagnosed Diseases Network25,26,27,28; genome sequencing is available in dbGaP (accession number: phs001232.v1.p1). Trio clinical exome sequencing was performed on Individual 2 and her parents at Blueprint Genetics (Seattle, WA; https://blueprintgenetics.com/). GeneMatcher (https://genematcher.org/), an online tool for connecting researchers and/or clinicians with an interest in the same gene24, was used to facilitate collaboration.

Research re-analysis of data from Individual 1 and his family was done through the NIH Undiagnosed Diseases Program (UDP). Briefly, sequencing reads were filtered for quality and aligned to human reference genome NCBI build 37 (hg19) using a pipeline developed by the NIH UDP, one based on NovoAlign (Novocraft Technologies, Petaling Jaya, Malaysia), and separately, a diploid aligner40 that was run on a commercial platform (Appistry Inc., St. Louis, MO). Variants were called with HaplotypeCaller and GenotypeGVCFs41,42,43. Variants were annotated using snpEff44 and a combination of publicly available data sources (gnomAD, ESP, and 1000Genomes) and internal cohort statistics. These annotations were utilized to create a list of rare, non-synonymous, start-gain/loss, stop-gain/loss, frameshift, canonical splice site variants, and intronic variants (±20 bp) that were consistent with homozygous recessive, compound heterozygous, X-linked or de novo dominant disease models. These variants were manually inspected using the Integrative Genomics Viewer (IGV) and checked for publicly available clinical or functional data in OMIM, HGMD, and PubMed. Variants were interpreted and prioritized based on the clinical relevance of the gene and the pathogenicity of the variants using the ACMG-AMP guidelines45. In the absence of candidate variants with unambiguous clinical relevance such as those in the majority of UDP cases and in this case in particular, variants were prioritized by inferred significance based on Mendelian consistency, population frequency, and predicted pathogenicity, coalesced with published biological and functional data of the genes.

All candidate variants were validated by Sanger sequencing using the primers indicated in Supplementary Table 4 on genomic DNA from peripheral blood (Family 1) or buccal swabs (Family 2). The Multiplex PCR Kit (206145, Qiagen, Germantown, MD) was used to conduct PCR amplification using the following conditions: 1 cycle of 95 °C for 15 min; 35 cycles of 94 °C for 30 s, 53 °C (Family 1) or 57 °C (Family 2) for 1 min 30 sec, and 72 °C for 1 min; followed by 1 cycle of 72 °C for 10 min. Excess primers and unincorporated nucleotides were enzymatically removed using the ExoSAP-IT PCR Product Cleanup Reagent (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA) according to the manufacturer’s specifications. Sequencing was performed by Macrogen (Rockville, MD) and sequences were analyzed using Sequencher (version 5.4.6—Build 46289, Gene Codes, Ann Arbor, MI).

Isolation and culture of patient cells

Primary fibroblasts from affected Individuals 1, 2, and 3 were cultured from a forearm skin biopsy. Unaffected primary fibroblasts GM00969 (2-year-old Caucasian female), GM01652 (11-year-old Caucasian female), and GM09503 (10-year-old Caucasian male) (Coriell Institute for Medical Research, Camden, NJ) and ATCC60235894 (neonatal male) (American Type Culture Collection, Manassas, VA) were used as unaffected controls. Fibroblasts were cultured in high glucose DMEM (11965092, Gibco/Thermo Fisher Scientific, Gaithersburg, MD) with 10% fetal bovine serum (FBS, 10082, Gibco/Thermo Fisher Scientific, Gaithersburg, MD), and 1× antibiotic-antimycotic (15240, Gibco/Thermo Fisher Scientific, Gaithersburg, MD) at 37 °C with 5% CO2.

Lymphoblastoid cell lines were established from the peripheral blood samples of Individual 2, her affected younger sister Individual 3, and her mother by transformation with Epstein-Barr virus (EBV)-containing supernatant through the Tissue Culture and Biobanking Shared Resource (TCBSR) at Georgetown Lombardi Comprehensive Cancer Center (Washington, DC) using standard methods46. Commercially available unaffected lymphoblastoid cell line E. H. IV (Elaine IV) (ATCC CCL-104, American Type Culture Collection, Manassas, VA) and the lymphoblastoid cell line established from the mother’s peripheral blood sample were used as unaffected controls. Lymphoblastoid cells were cultured in RPMI 1640 Medium with GlutaMAX Supplement (61870036, Gibco/Thermo Fisher Scientific, Gaithersburg, MD) containing 10% FBS (10082, Gibco/Thermo Fisher Scientific, Gaithersburg, MD), and 1× antibiotic-antimycotic (15240, Gibco/Thermo Fisher Scientific, Gaithersburg, MD) at 37 °C with 5% CO2.

RNA extraction and reverse transcription

Cells were homogenized using the QIAshredder (79654, Qiagen, Germantown, MD) and total RNA was extracted using the RNeasy Mini Kit (74106, Qiagen, Germantown, MD). Genomic DNA was removed by on-column DNase I digestion (79254, Qiagen, Germantown, MD).

Reverse transcription was performed using the Omniscript Reverse Transcription Kit (205111, Qiagen, Germantown, MD) using up to 2 μg total RNA per 20 μl reaction and 1 μM Oligo-dT primer (O4387-.1 ML, Sigma-Aldrich, St. Louis, MO) according to the manufacturer’s specifications.

Gene expression analysis

TaqMan Gene Expression Master Mix (4369016, Applied Biosystems/Thermo Fisher Scientific, Foster City, CA) was used with the 7500 Fast Real-Time PCR System (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA) for gene expression analysis. The following conditions were used for amplification: 1 cycle of 50 °C for 2 min for uracil-N-glycosylase incubation, followed by 1 cycle of 95 °C for 10 min for DNA polymerase activation, followed by 40 cycles of 95 °C for 15 sec and 60 °C for 1 min for PCR amplification.

The relative quantification of gene expression was calculated using the delta-delta Ct method47 using the 7500 Software version 2.3 (Applied Biosystems/Thermo Fisher Scientific, Foster City, CA). Expression of HPRT1 and POLR2A was used as the internal controls. All TaqMan assays used for gene expression analysis are presented in Supplementary Table 5.

Immunoblot analysis

Cells were lysed in RIPA Buffer (R0278, Sigma-Aldrich, St. Louis, MO) containing 1× Complete Ultra Protease Inhibitor Cocktail (05892970001, Roche/Sigma-Aldrich, St. Louis, MO) for 15 min on ice. The cysteine protease inhibitor N-Ethylmaleimide (E3876, Sigma-Aldrich, St. Louis, MO) was added to 20 mM to stabilize the lipidated form of the LC3/GABARAP subfamily members by inhibiting the processing activity of ATG4 cysteine proteases14. The samples were homogenized by sonication (Model 250 Digital Sonifier, Branson Ultrasonics, Danbury, CT) at 10% amplitude for 30 s on ice. Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA) was added, and samples were incubated at 95 °C for 5 min. Samples were resolved on a 7.5% (for ATG4D) or 15% (for in vitro GABARAPL1 priming assay) or 4–15% or 4–20% gradient (for all other targets) polyacrylamide gel and transferred to a PVDF membrane (IPFl00010, Millipore, Burlington, MA). Membranes were blocked using Odyssey Blocking Buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature and subsequently incubated with primary antibodies diluted in Odyssey Blocking Buffer with 0.1% Tween 20 overnight at 4 °C. After four 5-min washes with TBS-T (0.1% Tween 20 in Tris-buffered saline), membranes were incubated with IRDye-conjugated secondary antibodies (1:10,000, LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature. After four 5-min washes with TBS-T, two 5-min washes with TBS were performed to remove residual Tween 20. Membranes were imaged on the Odyssey CLx Infrared Imaging System and analyzed using the CLx Image Studio version 3.1 software (LI-COR Biosciences, Lincoln, NE). All blots were derived from the same experiment and were processed in parallel. All primary antibodies and dilutions used for immunoblotting are presented in Supplementary Table 6. All uncropped images of the data presented in Figs. 3, 4 are presented in Supplementary Figs. 710.

Immunoblot analysis to confirm total basal GABARAPL1 protein levels

Primary fibroblasts were treated with vehicle or Bafilomycin A1 for 3 h. Cell pellets were lysed using RIPA Lysis Buffer (sc-24948, Santa Cruz Biotechnology) supplemented with Complete Mini Protease Inhibitor Cocktail (11836153001, Roche). The cysteine protease inhibitor N-Ethylmaleimide (E3876, Sigma-Aldrich), which has been shown to inhibit ATG4 activity and stabilize lipidated GABARAP and GABARAPL114, was added to a final concentration of 20 mM. The Pierce BCA Protein Assay Kit (23225, Thermo Scientific) was used for total protein quantification, and 20 μg of protein was loaded on a 4–12% gradient Bolt Bis-Tris gel (Invitrogen) for separation. Gel was then transferred to a PVDF membrane (Bio-Rad Laboratories) and blocked with 2% milk solution before incubation overnight at 4 °C with primary antibodies. Primary antibodies were diluted with Odyssey® Blocking Buffer in PBS (LI-COR Biosciences). Membranes incubated with primary antibody were washed with 1× PBS-T (0.1% Tween 20) and incubated with the appropriate secondary antibody (goat anti-mouse IgG-horseradish peroxidase (HRP) and goat anti-rabbit IgG-HRP) (Santa Cruz Biotechnology) for 1 h. The SuperSignal™ West Femto Maximum Sensitivity Substrate (34096, Thermo Scientific) and ChemiDoc MP Imaging System (Bio-Rad Laboratories) were used to detect and visualize protein bands. Densitometry was performed using Image Lab Software (Bio-Rad Laboratories) to measure the relative protein of interest present by normalizing to loading control (β-actin).

Generation of an ATG4D overexpression cell line

The MGC Human ATG4D Sequence-Verified cDNA (MHS6278-202833433, GE Dharmacon, Lafayette, CO) was used to amplify the ATG4D ORF by PCR. PCR products were then cloned into the pENTR entry vector using the pENTR/D-TOPO Cloning Kit (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA). Single clones were Sanger sequenced to verify sequence integrity of the ATG4D ORF. LR recombination was performed using the Gateway LR Clonase II Enzyme Mix (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA) to recombine the ATG4D ORF into the pLenti6.3/V5-DEST destination vector (V53306, Invitrogen/Thermo Fisher Scientific, Carlsbad, CA). Single clones were Sanger sequenced to verify the recombination and sequence integrity of the ATG4D ORF.

The ViraPower Lentiviral Expression System (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA) was used to overexpress ATG4D. In brief, lentivirus was generated by transfecting 293FT cells with the pLenti6.3-ATG4D construct and the ViraPower Lentiviral Packaging Mix (K497500, Invitrogen/Thermo Fisher Scientific, Carlsbad, CA) using Lipofectamine 2000 Transfection Reagent (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA). Primary fibroblasts were transduced with the viral supernatant and stable cell lines were selected by antibiotic selection (2 μg/ml blasticidin) for 10 days. Overexpression of ATG4D mRNA and ATG4D protein were confirmed by quantitative PCR and immunoblot. TaqMan gene expression assays used for quantitative PCR and antibodies used for immunoblotting are presented in Supplementary Tables 5, 6, respectively.

Generation of an ATG4D-deficient HeLa cell line

CRISPR-Cas9 technology was used to generate an ATG4D-deficient HeLa cell line. In brief, a HeLa cell line with stable and constitutive Cas9 expression (SL503, GeneCopoeia, Rockville, MD) was transfected with three pGS-gRNA-Neo plasmids (GenScript, Piscataway, NJ) each containing a guide RNA, under the control of the U6 promoter, targeting ATG4D using Lipofectamine 2000 Transfection Reagent (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA) according to the manufacturer’s specifications. In parallel, the phU6-gRNA plasmid (#53188, Addgene, Watertown, MA) was transfected to serve as an empty vector control. Transfected cells were selected with 500 μg/ml Geneticin (10131035, Gibco/Thermo Fisher Scientific, Gaithersburg, MD) for 10 days and were maintained in high glucose DMEM (11965092, Gibco/Thermo Fisher Scientific, Gaithersburg, MD) with 100 μg/ml Geneticin (10131035, Gibco/Thermo Fisher Scientific, Gaithersburg, MD), 10% fetal bovine serum (FBS, 10082, Gibco/Thermo Fisher Scientific, Gaithersburg, MD), and 1× antibiotic-antimycotic (15240, Gibco/Thermo Fisher Scientific, Gaithersburg, MD) at 37 °C with 5% CO2 thereafter. The deficiency of ATG4D mRNA and ATG4D protein was confirmed by quantitative PCR and by immunoblot analysis, respectively (Supplementary Fig. 5A, B). The gRNA sequences are listed in Supplementary Table 7, TaqMan assay IDs used for quantitative PCR are listed in Supplementary Table 5, and antibodies used for immunoblotting are listed in Supplementary Table 6.

Generation of ATG4 tetra knockout rescue HeLa cell lines

The ATG4 tetra knockout and ATG5 knockout HeLa cell lines were gifts from Dr. Michael Lazarou (Biomedicine Discovery Institute, Monash University, Melbourne, Australia)18,48. Lentiviruses were generated to express WT or each ATG4D missense variant as described above. In brief, site-directed mutagenesis was performed to introduce ATG4D missense variants in the pENTR-ATG4D plasmid using the Q5 Site-Directed Mutagenesis Kit (E0554S, New England BioLabs, Ipswich, MA) and the primers listed in Supplementary Table 4. All constructs were verified by Sanger sequencing. LR recombination was performed using the Gateway LR Clonase II Enzyme Mix (Invitrogen/Thermo Fisher Scientific, Carlsbad, CA) to recombine the wildtype and variant ATG4D ORFs into the pLenti6.3/V5-DEST destination vector (V53306, Invitrogen/Thermo Fisher Scientific, Carlsbad, CA). Single clones were Sanger sequenced to verify the recombination and sequence integrity of the ATG4D ORF. Expression of V5-tagged ATG4D WT and variant proteins after viral transduction and antibiotic selection were confirmed by immunoblot. Antibodies used for immunoblotting are presented in Supplementary Table 6.

Transmission electron microscopy (TEM)

To assess the area and size of autophagosomes formed in response to the autophagy inducer Torin 1 and autophagy inhibitor Bafilomycin A1, cultured cells were treated with 100 nM of each inhibitor or vehicle (DMSO) for 3 h. Cells were then fixed using 2.5% glutaraldehyde and 1% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.4) at room temperature. After 30 min, the cells were scraped from the culture plate and centrifuged at 16,000 × g for 5 min; fixation was continued as a cell pellet for 1 h 30 min at room temperature and for a minimum of 48 h at 4 °C thereafter. Cell pellets were washed three times with 0.1 M cacodylate buffer, fixed with 1% OsO4 for 2 h, washed three times with 0.1 M cacodylate buffer, washed with water, and incubated in 1% uranyl acetate for 30 min. After fixation, the samples were subsequently dehydrated, embedded, sectioned, and stained for transmission electron microscopy (TEM) as previously described49.

Morphometric analysis of autophagosomes from TEM images

ImageJ was used to analyze the morphometry of autophagosomes in TEM images taken at 4,000×50. For each image, the cytoplasmic area was calculated by selecting the whole cell and the nucleus as regions of interest using the polygon tool, measuring the total area and the nuclear area, and then subtracting the nuclear area from the total area.

Autophagosomes were identified based on their distinctive morphology of cytoplasmic contents within double-membraned organelles31,51. Each autophagosome was selected as a region of interest using the polygon tool and the area was measured. Autophagosome area was assessed as the % cytoplasmic area per image and autophagosome size was assessed as the area of each individual autophagosome per sample.

in vitro GABARAPL1 priming assay

The N-terminal 6xHis-tagged pTrcHisB-ATG4B, pTrcHisB-ΔN63 ATG4D, pTrcHisB-GABARAPL1-myc, and pTrcHisB-GABARAPL1-G116A-myc bacterial expression plasmids were a gift from Dr. Jon D. Lane (University of Bristol, United Kingdom)15. Site-directed mutagenesis was performed to introduce patient variants into the pTrcHisB-ΔN63 ATG4D construct using the Q5 Site-Directed Mutagenesis Kit (E0554S, New England BioLabs, Ipswich, MA) and the primers used are listed in Supplementary Table 4. To perform site-directed mutagenesis for the c.1310_1328del variant, deletion of the relevant 19 bp was performed, followed by an insertion to mimic the predicted frameshift variant. All constructs were verified by Sanger sequencing.

Recombinant proteins were expressed in BL21-CodonPlus (DE3)-RIPL Competent Cells (230280, Agilent Technologies, Santa Clara, CA). A single colony was cultured in 10 ml LB media containing 100 μg/ml carbenicillin and 50 μg/ml chloramphenicol at 37 °C and 250 rpm until the OD600 measured 0.5-0.6. Each culture was expanded by the addition of 1 ml of the starter culture to 50 ml of LB media without antibiotics and cultured at 37 °C and 250 rpm until the OD600 measured 0.5-0.6. The cultures were then cooled to room temperature and induced with 0.2 mM IPTG at 16 °C and 225 rpm for 16 h.

Recombinant proteins were purified using TALON Spin Columns (Takara Bio, Mountain View, CA). Briefly, cells were collected by centrifugation at 3500 × g for 20 min at 4 °C. Lysates were prepared by the addition of 10 ml xTractor Buffer (HisTALON Buffer Set, Takara Bio, Mountain View, CA) containing 2 U/ml DNase I (4716728001, MilliporeSigma, Burlington, MA), 6 mM MgCl2, and 1 mM CaCl2. For the purification of 6xHis-ATG4B, 6xHis-GABARAPL-myc, and 6xHis-GABARAPL1-G116A-myc, ProteoGuard EDTA-free Protease Inhibitor Cocktail (Takara Bio, Mountain View, CA) was added according to the manufacturer’s specifications. For the purification of 6xHis-ΔN63 ATG4D, 40 μg/ml bestatin (10874515001, MilliporeSigma, Burlington, MA), 2 μg/ml leupeptin (11017101001, MilliporeSigma, Burlington, MA), 2 μg/ml aprotinin (10236624001, MilliporeSigma, Burlington, MA), 1 μg/ml pepstatin (10253286001, MilliporeSigma, Burlington, MA), and 75 μg/ml lysozyme (L1667, MilliporeSigma, Burlington, MA) were added. Cell pellets were gently resuspended and incubated with gentle shaking for 1 h at 4 °C. Following cell lysis, samples were sonicated at 20% amplitude for 6 × 10-s pulses on ice with 10 sec cooling in between each pulse. Lysates were clarified by centrifugation at 6000 × g for 40 min at 4 °C. Clarified lysates were then concentrated using an Amicon Ultra-15 Centrifugal Filter Unit (UFC901024, MilliporeSigma, Burlington, MA). The TALON Spin Columns (Takara Bio, Mountain View, CA) were equilibrated, and clarified lysates were added to the column according to the manufacturer’s specifications. Following sample binding, the column was washed three times with equilibration buffer (HisTALON Buffer Set, Takara Bio, Mountain View, CA) and an intermediate wash was performed with wash buffer (10 mM imidazole in Equilibration Buffer from the HisTALON Buffer Set, Takara Bio, Mountain View, CA) prior to elution. His-tagged proteins were eluted with 2 × 600 μl elution buffer (HisTALON Buffer Set, Takara Bio, Mountain View, CA). The 6xHis-ΔN63 ATG4D recombinant proteins were concentrated using an Amicon Ultra-0.5 Centrifugal Filter Unit (UFC501096, MilliporeSigma, Burlington, MA).

To assess the GABARAPL1 priming activity of each ATG4D variant, a previously described in vitro GABARAPL1 priming assay was performed15. Purified recombinant proteins were incubated for 0, 1, 2, 3, or 4 h at 37 °C in HEPES buffer (0.1% CHAPS, 10% (w/v) sucrose, 5 mM DTT, 2 mM EDTA, 50 mM HEPES, pH 7.4), and reactions were stopped by the addition of Laemmli Sample Buffer (Bio-Rad Laboratories, Hercules, CA) to 1× and samples were incubated at 95 °C for 5 min and resolved on a 15% polyacrylamide gel. Priming activity was analyzed by immunoblotting using the GABARAPL1 and myc antibodies noted in Supplementary Table 6.

GABARAPL1 priming rescue experiment

To assess the GABARAPL1 priming activity of each ATG4D missense variant, a previously described GABARAPL1 priming rescue experiment was performed18. In brief, ATG4 tetra knockout cell lines expressing V5-tagged wildtype or variant ATG4D were evaluated for their ability to prime GABARAPL1 in the presence or absence of 8 h Bafilomycin A1 treatment by immunoblot. The presence of lipidated GABARAPL1-II, which requires the formation of primed GABARAPL1-I from pro-GABARAPL1, was considered as successful priming by an ATG4 isoform. Antibodies used for immunoblotting are presented in Supplementary Table 6.

Statistical analyses

All statistical tests were performed using GraphPad Prism 8 Version 8.4.3. For experiments comparing two sets of data, a two-tailed Mann–Whitney U test was performed. For experiments involving the comparison of multiple sets of data, a one-way ANOVA or Kruskal–Wallis test was performed to compare relevant predefined dataset pairs and a Dunnett’s or Dunn’s multiple comparisons post hoc test was performed to correct for multiple comparisons, respectively. A p-value of less than 0.05 was considered statistically significant.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

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