BI-ALLELIC VARIANTS IN TM2D3 CAUSE A SEVERE SYNDROMIC NEURODEVELOPMENTAL DISORDER ASSOCIATED WITH ENDOPLASMIC RETICULUM AND MITOCHONDRIAL ABNORMALITIES.

Claudie Gabillard-Lefort, Caroline Silveira Martinez, Naïg Gueguen, Valérie Desquiret-Dumas, Méline Wery, Louis Legoff, Anne Guimier, Sophie Rondeau, Giulia Barcia, Christine Barnerias, et al.

► To cite this version:

Claudie Gabillard-Lefort, Caroline Silveira Martinez, Naïg Gueguen, Valérie Desquiret-Dumas, Méline Wery, et al. BI-ALLELIC VARIANTS IN TM2D3 CAUSE A SEVERE SYNDROMIC NEURODEVELOPMENTAL DISORDER ASSOCIATED WITH ENDOPLASMIC RETICULUM AND MITOCHONDRIAL ABNORMALITIES. American Journal of Human Genetics, 2025, 112 (7), pp.1711-1721. 10.1016/j.ajhg.2025.05.004. hal-05283235

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Distributed under a Creative Commons Attribution 4.0 International License
BI-ALLELIC VARIANTS IN TM2D3 CAUSE A SEVERE SYNDROMIC
NEURODEVELOPMENTAL DISORDER ASSOCIATED WITH
ENDOPLASMIC RETICULUM AND MITOCHONDRIAL ABNORMALITIES.

Authors

Claudie GABILLARD-LEFORT*¹, Caroline SILVEIRA MARTINEZ*¹, Naïg GUEGUEN¹,², Valérie DESQUIRET-DUMAS¹,², Méline WERY³, Louis LEGOFF⁴, Anne GUIMIER⁵, Sophie RONDEAU⁵, Giulia BARCIA⁵, Christine BARNERIAS⁶, Benjamin COGNE⁷, Thomas BESNARD⁷, Elsa LORINO⁸,⁹, Jessica DOUGLAS¹⁰, Olaf BODAMER¹⁰, Annalisa VETRO¹¹, Renzo GUERRINI¹¹, Simona BALESTRINI¹¹, Valerio CONTI¹¹, Laura SIRI¹², Arnaud CHEVROLLIER¹, Céline BRIS¹,⁴, Estelle COLIN¹,⁴, Vincent PROCACCIO¹,⁴, Delphine PRUNIER-MIREBEAU¹,², Guy LENAERS¹,¹³, Salim KHIATI¹, Mathilde NIZON*⁷ and Olivier R. BARIS*¹

*= These authors contributed equally

Affiliations:

¹University of Angers, MitoLab, Unité MITOVASC, UMR CNRS 6015, INSERM U1083, SFR ICAT,
University Hospital of Angers, Angers, France

²Departments of Biochemistry and Molecular Biology, University Hospital of Angers, Angers, France.

³University of Angers, SFR ICAT, Angers, France

⁴Department of Genetics, University Hospital of Angers, Angers, France

⁵Genomic Medicine Service for Rare Diseases, Necker Enfants Malades Hospital, Paris,
France.

⁶Pediatric neurology service, Necker Enfants Malades Hospital, Paris, France.

⁷University Hospital of Nantes, Genomic Medicine service, University of Nantes, CNRS, INSERM, Institut du thorax, Nantes, France

⁸Department of Pediatrics, University Hospital of Nantes, Nantes, France

⁹SMR pédiatrique ESEAN APF France handicap (Paediatric Rehabilitation Services), Nantes,
France

¹⁰Division of Genetics and Genomics, Department of Pediatrics, Boston Children's Hospital/Harvard Medical School, United States.

¹¹Department of Neuroscience and Medical Genetics, Meyer Children's Hospital IRCCS, Florence, Italy

¹²Unit of Child Neuropsychiatry, IRCCS Istituto Giannina Gaslini, Genova, Italy
**ABSTRACT (250 words)**

We identified via exome sequencing bi-allelic variants in *TM2D3* in four affected individuals from four unrelated families with overlapping clinical presentations, including microcephaly, severe global developmental delay with absent speech, autistic features, heart malformation and dysmorphic facial features. *TM2D3* encodes a transmembrane protein present in many tissues, with a higher abundance in the central nervous system, but little is known about its function and cell localization. Here, by using chemical and genetically encoded probes in SNB75 cells, we show that TM2D3 is an endoplasmic reticulum (ER) protein. Further analysis on SNB75 *TM2D3*-Knockout cells as well as skin fibroblasts from affected individuals harboring the recurrent c.503G>A, p.(Gly168Asp) allele revealed an impact of TM2D3 on ER-stress response, with dysregulated expression of *ATF4*, *HSPA5* and *DDIT3*. Transmission electron microscopy highlighted ER swelling as well as unexpected secondary mitochondrial alterations including increased length, cristae width and ER-mitochondria distance. To gain further insights on the pathomechanisms at play, we performed RNA sequencing from the fibroblasts of the three individuals harboring the p.(Gly168Asp) variant and four available parents, and disclosed 21 differentially expressed genes, including genes coding for extracellular matrix components involved in the migration of neuronal precursors. Altogether, these clinical and experimental data show that bi-allelic *TM2D3* variants underlie a severe syndromic neurodevelopmental disorder linked to exacerbated ER-stress sensitivity, secondary mitochondrial alterations and altered extracellular matrix gene expression.

<footnote n="13">¹³Department of Neurology, University Hospital of Angers, Angers, France</footnote>

<footnote>**Correspondence:** olivier.baris@univ-angers.fr</footnote>

<footnote>**KEYWORDS:** TM2D3, Neurodevelopmental disorder, Endoplasmic Reticulum, Mitochondria</footnote>
Neurodevelopmental disorders (NDDs) are a very heterogeneous group of embryologic and early-postnatal-onset conditions characterized by abnormalities in the development of the nervous system. They encompass autism spectrum disorders, developmental delay, intellectual disability, language disorders, attention deficit/hyperactivity disorders and epilepsy, alone or in combination. NDDs have a complex etiology, with inherited or *de novo* pathogenic variants often underlying the cause¹. Despite significant progress in the identification of the culprit genetic factors involved in these disorders through the emergence of exome and whole-genome sequencing², many affected individuals remain without molecular diagnosis and consequently adequate genetic counselling.

Herein, we identified four affected individuals, two males and two females (Figure 1 and Figure 2A), aged 5 to 24 years old, with overlapping clinical features, which are summarized in Table 1 and Supplemental Note. Antenatal features like intrauterine growth retardation, polyhydramnios or kidney malformation were noticed for three individuals, while all presented postnatal severe and global developmental delay. Three individuals began walking between 2 and 4 years, with wide-based gait or aided, and one individual could not walk at 5 years ½. Limb hypertonia was noticed in three individuals. They communicate through vocalizations, bi-syllabic words, and body language. All exhibited behavioral disorders including anxiety, periods of agitation and autistic features. No developmental regression was described. Focal seizures were present in two individuals, beginning at 22 months for individual 2 and at 2 months for individual 3. Individual 3 also had paroxysmal episodes of transient paresis suggestive of alternating hemiplegia beginning at 2 years ½. Postnatal microcephaly was present in all (OFC: from -2.5 SD to -5 SD). Brain magnetic resonance imaging (MRI) analyses in three individuals revealed small frontal lobes with global hypotrophy for Individual 1 and abnormal gyration for Individuals 2 and 3. Mild vermis hypoplasia was also noticed in Individual 3 and thick *corpus callosum* in Individual 2 (Figure S1). Individual 4 only had a CT-scan at 3 years showing a pineal cyst, which does not rule out frontal anomalies as well. There was no longitudinal significant change over the different MRI scans. Congenital extracerebral anomalies were present in all individuals including heart malformation (4/4: patent ductus arteriosus, polyvalvular dysplasia, ventricular septal defect), kidney malformation (2/4: horseshoe kidney), genital malformation (2/2 males: scrotal hypoplasia, cryptorchidism). Similar dysmorphic facial features were noticed in all
individuals including mainly mild coarse facial features, convex nasal ridge with low columella and pinched mouth.

The procedure and the cohort of donors was approved by the local French ethic committee « CPP Grand Ouest IV » (reference number: MESR DC-2017-2987). Informed consent was obtained for all individuals, probands and healthy parents. Exome sequencing revealed bi-allelic variants of *TM2D3* (MIM: 610014) in homozygous state in individual 3 and in compound heterozygous state in individuals 1, 2, and 4. Each parent, as well as the two healthy sisters of individual 2, was confirmed to be heterozygous for one of the *TM2D3* variants. Five variants were identified including two missense variants (*NM_078474.2:c.503G>A*, p.(Gly168Asp) and c.677C>T, p.(Thr226Met)), one nonsense variant (c.7G>T, p.(Gly3*)), one frameshift variant (c.153dup, p.(Val52Serfs*9)), and a 33 Kb deletion (chr15:102182681-102215940 [GRCh37]) encompassing all coding exons of the main *TM2D3* isoform (*NM_078474.2*, exons 1-6). All individuals carried at least a missense variant in either homozygous or compound heterozygous state with a loss-of-function variant. The p.(Gly168Asp), p.(Thr226Met), and p.(Gly3*) variants were present in the gnomAD database (v4.1.0) with a very rare allele frequency (1x10⁻⁵, 1.05x10⁻⁵, and 5.01x10⁻⁶ respectively, **Table 2**) and no homozygotes reported. The p.(Val52Serfs*9) variant was absent from gnomAD. Both p.(Gly168Asp) and p.(Thr226Met) affect highly conserved amino acids, that are intolerant to missense variants (**Figure 2B**) and predicted to be damaging or probably damaging according to PolyPhen2, SIFT, AlphaMissense, CADD and REVEL algorithms (**Table 2**). In addition, 3D modelling strongly suggests a severe disruption of the TM2D3 protein folding, in particular for the p.(Gly168Asp) variant (**Figure 2C**). Both p.(Gly3*) and p.(Val52Serfs*9) variants are predicted to result in nonsense-mediated mRNA decay. The recurrent variant c.503G>A is located at the first position of exon 5. In silico analysis using SpliceAl through MobiDetails³ did not predict a splicing defect. RNA was isolated from PAXgene blood of individual 1 and their parents, reverse transcribed into cDNA, and Sanger sequenced, confirming that the variant does not affect splicing.

TM2D3 is a transmembrane protein present in many tissues, with a higher abundance in the central nervous system. TM2D3 protein sequence shares homology with the β-amyloid peptide binding protein TM2D1, which sensitizes cells to β-amyloid toxicity⁴. To date, all
reported *TM2D3* variants have uncertain significance, although the c.206C>T, p.(Pro69Leu) variant was identified in one individual at heterozygous state with early-onset or atypical dementia (ClinVar accession number: VCV000870552.1), and the c.464C>T, p.(Pro155Leu) variant was associated at heterozygous state with a higher risk of developing Alzheimer's disease (MIM: 104300) through exome-wide association analysis in the Icelandic population (rs139709573)⁵,⁶. Two recent studies suggest that TM2D3 is involved in Notch signaling⁷,⁸. However, the subcellular localization of the protein remains unclear⁸, as the Hum-PLoc predictive tool⁹ locates the TM2D3 protein to the plasma membrane when using the full sequence (score of 1.66) or in mitochondria when using the first 50 amino acids (score of -0.19). To explore TM2D3 localization *in vitro*, we generated SNB75 *TM2D3* knockout cells using CRISPR/Cas9 (**Figure S2**) and transfected them either with a control GFP plasmid, or plasmids expressing wild-type (WT) or p.(Gly168Asp) TM2D3 canonical coding sequence (247 amino acids), or the short isoform of TM2D3 (221 amino acids) or a truncated form lacking the N-terminus predicted signal peptide (33 first amino acids), all fused to the GFP, in the presence of fluorescent probes for mitochondria or the endoplasmic reticulum (ER). While the control GFP pattern was diffuse, we observed a clear cytoplasmic pattern co-localized with the ER for both the WT and mutated TM2D3-GFP plasmids (**Figure 3A and Figure S3A**), left and middle panels, GFP ER-Tracker® + merge), and excluding a mitochondrial localization (**Figure 3A and Figure S3A**, right panels, TMRM + Merge). The short isoform showed the same ER localization pattern (**Figure S3A**). Interestingly, the GFP signal for the N-terminus truncated form of TM2D3 was diffuse, and the ER-co-localization was lost (**Figure S3A**). Moreover, co-transfection of the SNB75 cells with a plasmid encoding the predicted signal peptide of TM2D3 (N33-TM2D3-GFP), fused to GFP and an ER-tagged DsRed plasmid, confirmed the ER targeting (**Figure S3B**), further demonstrating that WT as well as the p.(Gly168Asp) TM2D3 are ER proteins.

Since ER is crucial for proper folding and modification of many proteins, and its dysfunction is often reflected by an intracellular stress involving ATF4, GRP78 and DDIT3, we explored the transcriptional regulation of these three markers in the context of TM2D3 absence or p.(Gly168Asp) variant. In *TM2D3-KO* SNB75 cells, we first observed a mild but significant down-regulation of all three transcripts under basal conditions compared to WT SNB75 cells,
whereas their expressions were dramatically increased upon tunicamycin treatment, a well-established ER stress inducer inhibiting protein N-linked glycosylation (Figure 3B). These expression patterns were partly observed in individuals' fibroblasts, as only HSPA5 (MIM: 138120) showed significantly decreased expression under basal conditions and was strongly increased upon tunicamycin treatment (Figure 3C and S4). To further demonstrate the involvement of TM2D3 in this altered ER stress response, we performed rescue experiments in TM2D3-KO SNB75 cells with plasmids encoding the short or long isoforms of TM2D3, as well as the p.(Gly168Asp) variant, and measured the abundance of GRP78 (protein encoded by HSPA5) by Western blot (Figure S5). SNB75 TM2D3-KO cells again showed a down-regulation of GRP78 under basal condition and increased accumulation upon tunicamycin treatment. Strikingly, transfection with the WT isoforms of TM2D3 restored GRP78 abundance to control values in both conditions. In contrast, expression of the p.(Gly168Asp) variant restored GRP78 levels under basal condition, and showed a tendency to increased accumulation compared to WT cells upon ER stress induction (p=0.09, not significant), suggesting a partial loss of function. Altogether, these results demonstrate that TM2D3 acts on the ER stress response, which is significantly exacerbated in individuals' cells. Further analyses of SNB75 TM2D3-KO cells and individuals' fibroblasts by transmission electron microscopy disclosed ER luminal swelling (Figure 4A-B, white arrows) and unexpected mitochondrial morphological changes such as longer mitochondria (Figure 4C and Figure S6), wider cristae (Figure 4D and Figure S6), and increased distance between mitochondria and the ER networks, compared to control or parental cells (Figure 4E and Figure S6). In addition, we observed an increased expression of mitochondrial fusion protein OPA1 in SNB75 TM2D3-KO cells (Figure S7), suggesting that these morphological changes are secondary to altered TM2D3 function. However, dosage of the enzymatic activities of the respiratory chain complexes and oxygen consumption measurements did not show abnormality in individuals 1 and 2 compared to control values (Table S1).

To gain further insights about the molecular pathomechanisms, we performed RNA sequencing from the fibroblasts of the three individuals harboring the p.(Gly168Asp) variant and four parents, and disclosed 21 differentially expressed genes (Figure S8A and Table 3, adjusted p-value ≤ 0.05, 5 genes overexpressed, 16 genes down-regulated, with fold-change
≥ ±1.5). Interestingly, gene ontology classification identified three extracellular matrix genes among the five that were overexpressed: *TNC* (MIM: 187380), *DPT* (MIM: 125597) and *COMP* (MIM: 600310) (Figure S8B).

Altogether, we describe here a severe syndromic neurodevelopmental disorder associated with microcephaly, malformations and dysmorphic facial features related to bi-allelic *TM2D3* variants. In our cohort, three individuals harbored a recurrent p.(Gly168Asp) variant in homozygous state, or compound with a nonsense or a frameshift variant. The fourth individual harbored a missense p.(Thr226Met) variant *in trans* with a large deletion including all coding exons of *TM2D3*. The p.(Thr226Met) variant has already been reported in homozygous state in two relatives from a consanguineous family¹⁰. One individual had mild intellectual disability, aggressive behavior, and dental caries, and the other one had severe intellectual disability, delayed language and walking, spasticity, and aggressive behavior. Further rare *TM2D3* variants were associated with Alzheimer disease and frontotemporal dementia but were not related to a developmental disorder⁵,⁶.

*TM2D3* is an evolutionary conserved gene located on chromosome 15q26.3, which encodes 6 isoforms in humans. The conventional isoform, corresponding to the NM_078474.3 transcript, has a double-pass transmembrane TM2 domain and is ubiquitously expressed, based on the Genotype-Tissue Expression consortium (GTEx) data, including most brain regions¹¹. Alternative *TM2D3* transcripts are also expressed across diverse human tissues⁵.

To date, most knowledge about TM2D3 originates from studies in *Drosophila melanogaster*. Modeling of the human p.(Pro155Leu) variant in *amx*, the Drosophila *TM2D3* ortholog, revealed a loss-of-function effect causing embryonic phenotypes associated with altered Notch signaling⁵. Using knockout models, Salazar and colleagues showed that *TM2D* genes (*TM2D1*, *TM2D2* and *amx*) have overlapping functions in the Notch pathway and work together⁷. More recently, it was demonstrated that TM2D3 is involved in the cell surface presentation of Notch receptors both in Drosophila cells and embryonic *Tm2d3* knockout mouse cells⁸. Moreover, the data gathered by the International Mouse Phenotyping Consortium strongly suggests that homozygous *Tm2d3* knockout is lethal, which is consistent with the absence of individuals harboring bi-allelic complete loss-of-function *TM2D3*.
variants. We thus hypothesize that both missense variants we identified behave as hypomorphic alleles partially maintaining the protein function.

Among the clinical manifestations of the *TM2D3*-linked presentations we observed, microcephaly occurred in all individuals, testifying abnormal brain development, deriving from imbalanced generation, growth or survival of neural progenitor cells¹². ER stress, for which we identified an exacerbated response in individuals’ fibroblasts and *TM2D3*-KO SNB75 cells, was already reported in other individuals with microcephaly¹³. Indeed, proliferation of neuronal precursors during corticogenesis requires an increased synthesis flow of protein properly folded and maturated in the ER. In this respect, accumulation of misfolded proteins in the ER lumen triggers the unfolded protein response (UPR), which ultimately leads to their degradation via the ubiquitin-proteasome pathway. The UPR response is crucial during neurogenesis and therefore tightly regulated¹⁴, or leading to apoptosis when the ER stress is sustained and unbalanced, thus interfering with physiological neurogenesis. Such detrimental chain of events involving the UPR chaperone GRP78 is responsible for Zika virus (ZIKV)-associated congenital microcephaly¹⁵, which is due to impaired indirect neurogenesis and increased apoptosis in the developing brain¹⁶,¹⁷.

We further observed secondary mitochondrial alterations in individuals’ fibroblasts and SNB75 *TM2D3*-KO cells, particularly increased mitochondrial length. Mitochondrial dynamics is crucial for neurons to maintain a constant transport of mitochondria between cell soma and synapse. Several *DNM1L* (MIM: 603850) pathogenic variants result in enlarged mitochondria and encephalopathy with microcephaly as we disclosed here¹⁸,¹⁹, emphasizing the involvement of an adequate mitochondrial morphology for proper brain development.

In our cell models, we also observed an increased distance between mitochondria and the ER, which might affect specialized structures called Mitochondria Endoplasmic Reticulum Contact sites (MERCs). MERCs are important for a wide array of cellular functions, including lipid trafficking, calcium homeostasis, apoptosis or autophagy²⁰. MERCs destabilization results in altered inter-organellar communication and pathological conditions, as evidenced in mouse expressing the organelle spacer FATE1²¹. Pathological variants in genes coding for proteins involved in MERCs are involved in neurodevelopmental disorders²²,²³. MERCs also control mitochondrial morphology through the recruitment of DNM1L²⁴ to promote
mitochondrial fission, further suggesting that unbalanced mitochondrial elongation in *TM2D3*-KO cells and fibroblasts results from impaired MERCs and that this detrimental chain of events is involved in the altered brain development observed in our cohort of individuals.

Among the genes overexpressed in the three individuals with the p.(Gly168Asp) allele, three encode components of the extracellular matrix (Tenascin C, Dermatopontin and Cartilage oligomeric matrix protein). Tenascin C (TNC) is a glycoprotein, which gene is widely expressed during embryogenesis and in a tissue-restricted fashion during adult life²⁵. It modulates cell adhesion to the matrix²⁶ and is involved in the development of the central nervous system²⁷, where it contributes to the migration and proliferation of neural precursor cells²⁸,²⁹ and regulates multiple functions of microglial cells, such as phagocytosis and chemotaxis, through interaction with the Toll-Like Receptor 4³⁰ (TLR4), which is down-regulated in individuals with *TM2D3* variants. In parallel, we identified a down-regulation of the Inka Box Actin Regulator 2 gene (*INKA2*, MIM: 620403), which encodes an evolutionary conserved protein predominantly expressed in the central nervous system³¹. INKA2 controls actin polymerization and the development of dendritic spines through the inhibition of the serine/threonine-protein PAK4³². Last, although we did not identify clear bonds between the 21 differentially expressed genes and the Notch signaling pathway, we cannot exclude its involvement in the phenotype of our individuals, given its pleiotropic roles in organ development.

In conclusion, we describe four individuals carrying biallelic *TM2D3* variants, which induce a primary alteration of the ER-stress response and secondary mitochondrial alterations as well as dysregulated expression of genes involved in neuronal migration and their extracellular matrix composition. Our results indicate that pathogenic variants in *TM2D3* cause a severe syndromic neurodevelopmental recessive disorder associated with microcephaly, facial features, and multiorgan involvement, possibly by impairing different pathways involved in key developmental processes. These findings demonstrate the role of *TM2D3* in human pathology and provide insights for understanding the molecular mechanisms involved in TM2D3 dysfunction.
## DATA AND CODE AVAILABILITY

Identified *TM2D3* variants have been deposited to ClinVar (SCV005387825 to SCV005387828). The complete raw dataset for the RNA sequencing experiment is available in the BioProject Database (BioProject ID: PRJNA1185456).

## ACKNOWLEDGEMENTS

The authors thank the individuals and their families for their participation in this study. We also thank the Bioinformatics Core Facility of Nantes BiRD, member of Biogenouest, Institut Français de Bioinformatique (IFB) (ANR-11-INBS-0013) for the use of its resources and its technical support. We thank Mireille Wertheimer, Dorine Bouvier and Louane Daguin for excellent technical help. We also thank Florence Manero and Rodolphe Perrot from the SCIAM microscopy core facility (SFR ICAT). This work was supported by Pays de la Loire region and University of Angers (INGEMICA, PULSAR program), Agence nationale de la recherche (ANR-20-CE92-0020) and Association Française contre les Myopathies (AFM DYNAMYO #25002 and #25034). RG, AV, VC and SB are supported by funding of the Current Research 2023 by the Italian Ministry of Health.

## DECLARATION OF INTEREST

The authors declare no conflicts of interest related to the contents of this manuscript.

## AUTHOR CONTRIBUTIONS

**Experiments:** CGL, CSM, NG, VDD, AC, CB, SK; **Data analysis:** CGL, CSM, NG, VDD, MW, LL, BC, TB, AC, CB, EC, VP, DPM, GL, SK, MN, ORB; **Individuals recruitment and sample collection:** AG, SR, GB, CB, EL, JD, OB, AV, RG, SB, VC, LS, MN; **First manuscript draft:** CGL, CSM, MN, ORB; **Final manuscript reviewing, editing and approval:** all authors; **Funding:** RG, AV, VC, SB, ORB.
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FIGURE TITLES AND LEGENDS

**Figure 1:** *Clinical photographs of the four individuals.* Individual 1 aged 7 months and 4 1/2 years. Individual 2 aged 3 years 1/2, 4 years and 6 years. Individual 3 aged 20 month and 7 1/2 years. Individual 4 aged 3 years and 24 years. Note the similar dysmorphic features and the evolution of dysmorphism during childhood. Mouth is small and pinched in infancy with thin lips, nose is convex with low columella, and eyebrows are thin.

**Figure 2:** *The p.(Gly168Asp) variant of TM2D3 is in a conserved region and not well tolerated.* A: Pedigrees of the four affected individuals with bi-allelic, likely pathogenic TM2D3 variants. B: Sequence alignments showing the conserved amino acids positions of TM2D3 in multiple species. Tolerances of the p.(Gly168Asp) and p.(Thr226Met) variants were assessed using MetaDome. The p.(Gly168Asp) is less tolerated than the p.(Thr226Met) variant. C: 3D modelling of TM2D3 WT and the two missense variants identified in our study were generated using AlphaFold. The p.(Gly168Asp) variant induces a drastic conformational change compared to WT.

**Figure 3:** *TM2D3 protein is localized in endoplasmic reticulum and involved in ER-stress response to Tunicamycin.* A: SNB75 TM2D3-KO cells were transfected either with a plasmid carrying GFP, or TM2D3-WT/GFP, and incubated with TMRM and ER-tracker® dyes. Images were obtained by confocal microscopy and fluorescence spectrums were obtained using ImageJ®. Representative images of 4 independent experiments. Scale bar: 20µm B: Fold change of ER stress markers ATF4, HSPA5 and DDIT3 in SNB75 cells WT (white) or TM2D3_KO (dark) after 24h Tunicamycin incubation (1µg/ml). C: Fold change of ER stress markers ATF4, HSPA5 and DDIT3 in fibroblasts from parents (P, white) or individuals carrying TM2D3 variants (I, dark) after 24h Tunicamycin incubation (1µg/ml). n=3 biological replicates for each individual, and healthy parent *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001

**Figure 4:** *TM2D3 variants are associated with increased mitochondrial length, cristae width and ER-Mitochondria distance.* A: Electronic microscopy images from SNB75 TM2D3 WT and KO cells. Images are representative of mitochondria analyzed from 3 independent cells for each condition. Scale bar: 200nm. Arrows represent swelled ER. B: Electronic microscopy images from individuals' and parents' primary skin fibroblasts. Images are representative of
mitochondria analyzed from 3 independent cells for each individual. Scale bar: 200nm (left) and 100nm (right). Arrows represent swelled ER. C, D and E: Mitochondrial length, cristae width and ER-Mitochondria distance are increased in SNB75 TM2D3_KO and in the individuals. Mean of mitochondrial length, cristae width and ER-mitochondria distance were calculated from at least 50 different mitochondria or ER-mitochondria contacts per cell, n=3 cells per condition. For C, D and E: statistical analysis was performed using unpaired t tests, \*p<0.05, \*\*\*p<0.0001

## TABLE TITLES AND LEGENDS

**Table 1: Clinical features of the four individuals with TM2D3 variants** (GeneBank: NM_078474.2). IUGR: intrauterine growth retardation, OFC: occipital-frontal circumference, SD: standard deviation

**Table 2: Predictive analysis of the different TM2D3 variants found in our study.** Abbreviations: NR: Not Reported, P: Prediction, D: Damaging, PD: Probably Damaging, U: Uncertain.

**Table 3: Differentially expressed genes in individuals compared to the parents.** Abbreviations: NF-κB: Nuclear Factor kappa-light-chain-enhancer of activated B cells, ERK: Extracellular Signal-Regulated Kinase, MAP: Mitogen-Activated Protein, HIF-1α: Hypoxia-Inducible Factor 1-alpha, Th2 cell: T-helper type 2 cell, WNT: Wingless/Integrated, mTOR: Mechanistic Target of Rapamycin, GTPase: Guanosine Triphosphatase
<table><caption>**Table 1**</caption><thead><tr><th>Affected individuals</th><th>Individual 1</th><th>Individual 2</th><th>Individual 3</th><th>Individual 4</th></tr></thead><tbody><tr><td>Allele 1</td><td>c.503G&gt;A,<br>p.(Gly168Asp)</td><td>c.503G&gt;A,<br>p.(Gly168Asp)</td><td>c.503G&gt;A,<br>p.(Gly168Asp)</td><td>c.677C&gt;T, p.(Thr226Met)</td></tr><tr><td>Allele 2</td><td>c.7G&gt;T, p.(Gly3*)</td><td>c.153dup,<br>p.(Val52Serfs*9)</td><td>c.503G&gt;A,<br>p.(Gly168Asp)</td><td>Del 15q26.3 (33 Kb)</td></tr><tr><td>Origin</td><td>France</td><td>France</td><td>Italy</td><td>United States</td></tr><tr><td>Consanguinity</td><td>No</td><td>No</td><td>No</td><td>No</td></tr><tr><td>Sex</td><td>Male</td><td>Male</td><td>Female</td><td>Female</td></tr><tr><td>Years at last examination</td><td>6<sup>1/2</sup></td><td>6</td><td>5<sup>1/2</sup></td><td>26</td></tr><tr><td>Prenatal history</td><td>IUGR</td><td>IUGR, kidney<br>malformation,<br>polyvalvular dysplasia</td><td>Polyhydramnios, kidney<br>malformation</td><td>Decreased fetal activity</td></tr><tr><td>Birth data (term, weight,<br>height, OFC)</td><td>38 WG, 1870g, 46 cm,<br>32cm</td><td>35 WG, 1780g, 41 cm,<br>30 cm</td><td>36 WG, 2720g, NA, NA</td><td>36 WG, 25th p, 50th p,<br>10th p</td></tr><tr><td>Height at last examination</td><td>-3 SD</td><td>-3.3 SD</td><td>+1.3 SD</td><td>+0.3 SD</td></tr><tr><td>Weight at last examination</td><td>-2.5 SD</td><td>-2.5 SD</td><td>-0.5 SD</td><td>-1.8 SD</td></tr><tr><td>OFC at last examination</td><td>-5 SD</td><td>-3.6 SD</td><td>-2.5 SD</td><td>-4 SD</td></tr><tr><td>Developmental delay</td><td>Severe</td><td>Severe</td><td>Severe</td><td>Severe</td></tr><tr><td>Age of walking</td><td>Not achieved</td><td>With support at 4 years</td><td>With support at 3 years</td><td>Achieved at 2 years with<br>wide-based gait</td></tr><tr><td>Language</td><td>Vocalization</td><td>Vocalization</td><td>Vocalization, bi-syllabic<br>words</td><td>Vocalization</td></tr><tr><td>Neurological exam</td><td>Hypertonia, dystonia</td><td>Hypertonia, dystonia</td><td>Hemiplegic episodes</td><td>Hypertonia</td></tr><tr><td>Behavior</td><td>Anxiety, agitation,<br>relational disorder</td><td>Anxiety, autistic features</td><td>Agitation</td><td>Anxiety, agitation,<br>autistic features</td></tr><tr><td>Seizures</td><td>No</td><td>Yes, at 1 year 1/2</td><td>Yes, at 2 months with<br>alternating hemiplegia</td><td>No</td></tr><tr><td>Brain imaging</td><td>Global hypotrophy with<br>small frontal lobes at 18<br>months and 3 years 1/2</td><td>Small frontal lobes and<br>abnormal gyration, thick<br>corpus callosum at 5<br>years</td><td>Abnormal frontal<br>folding, vermis<br>hypoplasia at 1 year and<br>5 years</td><td>Pineal cyst at 3 years<br>(CT-scan only)</td></tr><tr><td>Abdominal ultrasound</td><td>Mild pyelocaliceal<br>dilatation</td><td>Horseshoe kidney</td><td>Horseshoe kidney</td><td>Normal</td></tr><tr><td>Cardiac ultrasound</td><td>Patent ductus<br>arteriosus, patent<br>foramen ovale</td><td>Polyvalvular dysplasia,<br>ventricular septal defect</td><td>Three ventricular septal<br>defects, vena cava<br>duplication</td><td>Ventricular septal defect</td></tr><tr><td>Ophtalmologic features</td><td>Hypermetropia</td><td>Hypermetropia,<br>strabismus</td><td>Normal</td><td>Strabism, legal blindness</td></tr><tr><td>Reproductive organs</td><td>Scrotal hypoplasia</td><td>Scrotal hypoplasia,<br>cryptorchidism</td><td>-</td><td>-</td></tr></tbody></table>
<table><caption>**Table 2**</caption><thead><tr><th rowspan="2">Individual</th><th rowspan="2">Variant</th><th rowspan="2">Variant Type</th><th rowspan="2">GnomAD allele frequency</th><th colspan="2">SIFT</th><th colspan="2">Polyphen-2</th><th colspan="2">Alpha Missense</th><th colspan="2">CADD</th><th colspan="2">REVEL</th></tr><tr><th>Score</th><th>P</th><th>Score</th><th>P</th><th>Score</th><th>P</th><th>Score</th><th>P</th><th>Score</th><th>P</th></tr></thead><tbody><tr><td>1, 2, 3</td><td>p.(Gly168Asp)</td><td>Missense</td><td>1x10<sup>-5</sup></td><td>0,02</td><td>D</td><td>1</td><td>PD</td><td>0,987</td><td>D</td><td>36</td><td>D</td><td>0,57</td><td>U</td></tr><tr><td>1</td><td>p.(Gly3*)</td><td>Nonsense</td><td>5.64x10<sup>-6</sup></td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td></tr><tr><td>2</td><td>p.(Val52Serfs*9)</td><td>Frameshift</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td><td>NR</td></tr><tr><td>4</td><td>p.(Thr226Met)</td><td>Missense</td><td>1.05x10<sup>-5</sup></td><td>0</td><td>D</td><td>1</td><td>PD</td><td>0,89</td><td>D</td><td>30</td><td>D</td><td>0,72</td><td>D</td></tr></tbody></table>
<table><caption>**Table 3**</caption><thead><tr><th colspan="4">Up-regulated genes</th></tr><tr><th>Name</th><th>Log2 FC</th><th>p value</th><th>Associated Pathway</th></tr></thead><tbody><tr><td>EYA2</td><td>3.632</td><td>7.220E-05</td><td>DNA Double Strand Break Response</td></tr><tr><td>DPT</td><td>2.877</td><td>1.104E-06</td><td>Cell adhesion, collagen fibril organization</td></tr><tr><td>COMP</td><td>1.714</td><td>3.273E-11</td><td>Extracellular Matrix organization, focal adhesion</td></tr><tr><td>TNC</td><td>1.117</td><td>1.059E-07</td><td>Integrin cell surface interaction, focal adhesion</td></tr><tr><td>LRRC20</td><td>0.733</td><td>6.305E-05</td><td>Innate immune response</td></tr></tbody><thead><tr><th colspan="4">Down-regulated genes</th></tr><tr><th>Name</th><th>Log2 FC</th><th>p value</th><th>Associated Pathway</th></tr></thead><tbody><tr><td>ARHGDIB</td><td>-3.800</td><td>3.948E-06</td><td>RhoA activity, stress induction of heat shock proteins</td></tr><tr><td>TLR4</td><td>-3.283</td><td>1.218E-09</td><td>NF-κB activation, immune response</td></tr><tr><td>EBF2</td><td>-3.184</td><td>2.925E-06</td><td>Adipogenesis, nervous system development</td></tr><tr><td>KRTAP1-1</td><td>-2.681</td><td>8.410E-05</td><td>Keratinization, nervous system development</td></tr><tr><td>PLEKHA6</td><td>-2.066</td><td>1.039E-04</td><td>Lipid and phospholipid metabolism</td></tr><tr><td>KRT19</td><td>-1.994</td><td>8.507E-05</td><td>Cytoskeleton remodeling, nervous system development</td></tr><tr><td>NEK10</td><td>-1.974</td><td>2.842E-05</td><td>ERK1/2 and MAP kinase activity regulation</td></tr><tr><td>INKA2</td><td>-1.781</td><td>7.768E-06</td><td>Protein kinase binding</td></tr><tr><td>FCRLA</td><td>-1.661</td><td>9.193E-06</td><td>Cell differentiation, immune response</td></tr><tr><td>SERPINB2</td><td>-1.512</td><td>7.495E-05</td><td>Blood clotting cascade, cytokine signaling in immune system</td></tr><tr><td>BHLHE41</td><td>-1.433</td><td>3.134E-07</td><td>HIF-1α network, Th2 differentiation</td></tr><tr><td>CARMIL1</td><td>-1.105</td><td>5.692E-06</td><td>Hemostasis</td></tr><tr><td>PAPSS2</td><td>-1.016</td><td>3.989E-06</td><td>Proteoglycan biosynthesis, sulfate activation for sulfonation</td></tr><tr><td>MYO1E</td><td>-1.005</td><td>3.559E-05</td><td>Epithelial adherens junctions</td></tr><tr><td>LGR4</td><td>-0.782</td><td>2.668E-05</td><td>WNT signaling, mTOR signaling</td></tr><tr><td>CPEB2</td><td>-0.691</td><td>2.422E-05</td><td>Negative regulation of GTPase activity, response to oxidative stress</td></tr></tbody></table>
<figure><img src="image_2.png" /></figure>

## Graphical abstract

## eTOC Blurb

TM2D3 is present in many tissues, but its function and cell localization are not fully elucidated. Gabillard-Lefort and colleagues report *TM2D3* variants that are associated with a syndromic neurodevelopmental disorder. They identified TM2D3 as an endoplasmic reticulum (ER) protein, which mutations leads to exacerbated ER-stress response and secondary mitochondrial alterations.
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>Figure 3</figcaption></figure>
A.

WT

<figure><img src="image_8.png" /></figure>

TM2D3_KO

<figure><img src="image_9.png" /></figure>

B.

I-1 (Family 1)

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

II-1 (Family 1)

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

C.

<figure><img src="image_14.png" /></figure>

D.

<figure><img src="image_15.png" /></figure>

E.

<figure><img src="image_16.png" /><figcaption>Figure 4</figcaption></figure>
# Supplemental Note

## Supplemental Note: Clinical Data

### Subjects

Clinical information was obtained from individuals with *TM2D3* bi-allelic pathogenic variants from France (Centre Hospitalier Universitaire Nantes, Institut Imagine Hôpital Necker-Enfants Malades), Italy (Meyer Children's Hospital IRCSS) and the United States (Boston Children's Hospital) and put together via Genematcher¹. Individuals were clinically assessed by a geneticist and a pediatrician or child neurologist. The study has been approved by the local ethics committee “CPP Ouest IV” (Reference number MESR DC-2017-2987). Written informed consents were obtained for genetic testing, skin biopsy for functional studies and use of photographs.

### Clinical data

**Individual 1** is the first boy of a non-consanguineous healthy couple with no family history. Intra-uterine growth retardation was noted early during pregnancy. He was born at 38 WG by caesarian section due to abnormal fetal heart rate. Growth measurements at birth were: weight 1970g (<< 3rd p), height 46 cm (3rd p), OFC 32 cm (3rd p).

At birth, complete II-III syndactyly was noted on both feet, as well as sacral dimple. Cranial, and medullar sonography was normal. Abdominal sonography showed a mild pyelocaliceal dilatation. Heart sonography revealed small patent ductus arteriosus and patent foramen oval.

At 3 months old, examination showed axial hypotonia, limb hypertonia and microcephaly (OFC -2.5 SD). Complete II-III foot syndactyly was associated with partial bilateral III-IV hand syndactyly and unique palmar transverse crease. Nasal reflux, superficial polypnea and scrotal hypoplasia were also noticed. Dysmorphic features included plagiocephaly, microstomia and thin lips.

At 5 years ½, the boy had severe psychomotor retardation, behavioral disorder, and growth retardation: height was 100cm (-2.5 SD), weight 15 Kg (-2 SD), OFC 44 cm (-6 SD). He could hold his head at 4 months, roll over at 18 months, be on crawling position at 2 years ½, and stand up with support at 5 years. At 5 years ½, language expression was limited to cries and vocalizations. He could catch something with palmar grip. He could smile, and make visual contact.

His behavioral disorder included anxiety with bruxism, depressive episodes, and periods of agitation with inconsolable crying mainly at sleeping, relational disorder, auto-aggressiveness, sleeping disorder with nocturnal awakenings. Treatment with Risperidone, Alimemazine and Melatonin proved effective for the sleeping disorder, crying episodes and auto-aggressiveness. Parents also noticed insensitivity to pain but he is very sensitive to
touching and massages. A spontaneously favorable episode of alopecia occurred at the age of 4 years.

Neurological examinations showed axial hypotonia, limb hypertonia with dystonic movements. Progressive and postnatal microcephaly was noticed with OFC 44 cm at 5 years ½ (-6 SD).

Seizures were suspected at 5 years but ruled out by 24-hour electroencephalogram. Brain MRI at 18 months and 3 years ½ showed global hypotrophy predominant on frontal lobes. Spinal MRI was normal.

Furthermore, ophthalmological examination revealed Claude-Bernard Horner syndrome at 1 year, and bilateral papillary drusen at 3 years with no consequence, associated with hypermetropia.

He had no hearing impairment. He had sleep apnea syndrome and laryngomalacia, with chronic rhinitis.

He had small teeth with eruption delay (1st tooth at 18 months), and fragile enamel.

Skin was very sensitive and marbled, with xosis, dried hair and normal nails.

He could eat mixed and was treated for constipation.

Genetics investigations showed normal array CGH and 7-dehydrocholesterol dosage was normal. Singleton exome sequencing revealed compound heterozygous variants in *TM2D3*: p.(Gly168Asp) and p.(Gly3*).

**Individual 2** is a 6 years' old boy, born to non-consanguineous healthy parents, after a dichorionic diamniotic twin pregnancy. There is no family history of neurodevelopmental disorders. The twin is a healthy girl.

During pregnancy, intra-uterine growth retardation was noted, associated with a horseshoe kidney and cardiac defects including valvular dysplasia, tricuspid and mitral regurgitation.

Twins were born at 35 WG by caesarian section due to growth retardation on the boy.

His birth parameters were: weight: 1780g (3rd p), height 41 cm (1st p), OFC 30cm (4th p) and he presented with bilateral cryptorchidism. Postnatal echocardiography confirmed polyvalvular dysplasia associated to ventricular septal defect and abdominal ultrasound confirmed horseshoe kidney.

At 7 months old, microcephaly (OFC at -2.5 DS), plagiocephaly, axial hypotonia and limb hypertonia were noted at examination. He underwent cardiac surgery at 12 months.

Epilepsy started at 22 months with generalized seizures first treated by sodium valproate then more focal seizures and lamotrigine was added several months later but poorly effective.

He had a severe neurodevelopmental disorder with delayed motor development: could sit at 12 months, walk with support at 4 years, and was nonverbal.

He had behavioral disorder with anxiety, autistic features, interaction disorder, auto-aggressiveness, and bruxism. He was very sensitive to noise. He presented with feeding and
mastication difficulties with sometimes food going down the wrong way. Progressive microcephaly and growth retardation were noted.

At 6 years, growth measurements were: height: 99 cm (-3.3 DS), weight: 15 kg (-2.2 DS), OFC: 47.5 cm (-3.6 DS). He could walk unaided at 6 years but had no language. He communicates and has contact by vocalization, cries, can point at a person or an object. He could catch objects or food with palmar grip. He can smile. There was no regression. Neurological examination revealed limb hypertonia and dystonia. Brain MRI revealed small frontal lobes with abnormal gyration, short and thick corpus callosum and enlarged bifrontal subarachnoid spaces.

Ophthalmological examination first revealed bilateral papillary pallor at 7 month, but was later controlled as normal. He wears glasses for hypermetropia and strabismus.

He had small teeth with eruption delay (1st tooth around 12-14 months).

Until 4 years old, he had recurrent pulmonary infections and chronic bronchitis with asthma.

He had xosis with very sensitive skin and slow-growing nails.

He had scoliosis and dysmorphic features including downslanting palpebral fissures, flat face, thin nose, plagiocephaly, pointed chin, small teeth, dysplastic ears, thin superior lip, and slight bilateral II-III toe syndactyly.

Array-CGH only revealed two benign CNVs: Xp22.12 dup (337 kb) and Xp22.31 dup (375 kb) both inherited from a healthy mother who had in turn inherited them from her asymptomatic father. Sequencing of the FBN1 and RAS pathway genes was unrevealing. Trio exome sequencing revealed compound heterozygous variants in TM2D3: p.(Gly168Asp) inherited from the mother and p.(Val52Serfs*9) inherited from the father.

**Individual 3** is a 5 years and 6 months old girl, first-born to non-consanguineous healthy parents with no family history of neuropsychiatric or congenital abnormalities. Pregnancy was complicated by threatened preterm labor at 33 weeks of gestation. Prenatal ultrasound detected polyhydramnios, horseshoe kidney and a complex cardiac malformation with 3 small ventricular septal defects (VSD) and persistent left superior vena cava. Delivery was by caesarean section at 36 weeks of gestation. Birth weight was 2720 g (50th p) and Apgar score was 4-7-8.

At birth the girl showed axial hypotonia and required mechanical ventilation (CPAP) because of bradycardia (80 bpm) and low oxygen saturation (O2sat 52-65%). Feeding difficulties with episodes of desaturation requiring mechanical ventilation and treatment with furosemide, were also observed since day 7.

Dysmorphic features were noticed, including sparse eyebrows, thin lips, mild mandibular hypoplasia, low set and posteriorly rotated ears, and bilateral II-III toe syndactyly, the latter also present in her mother.

Echocardiography and CT angiography better characterized the cardiopathy that featured 3 small VSDs, open ductus arteriosus with massive right-to-left shunt, persistent left superior
vena cava, embryologic remnant of the primitive pulmonary vein, and pulmonary hypertension, requiring surgery at age 1 month.

At age 2 months, paroxysmal episodes appeared with hypomotor behavior with reduced response to stimuli, hypotonia, at times stiffness, pallor and oral automatisms, occurring approximately twice per month. These episodes had the characteristics of early focal seizures. Interictal EEG recordings showed multifocal epileptiform abnormalities mainly during sleep. She was initially started on levetiracetam which was ineffective and led to behavioral deterioration; sodium valproate was subsequently added with initially complete seizure control but then her paroxysmal events recurred, and valproate was withdrawn due to liver derangement. She tried additional treatments over time including lamotrigine (ineffective), clonazepam (partially effective), carbamazepine (ineffective), lacosamide (partially effective), topiramate (tried for a few days, led to behavioral difficulties).

At age 2 years 6 months, paroxysmal episodes of transient paresis involving either side of the body started to occur, sometimes associated with dystonic posturing, resolving during sleep, which raised the suspect of Alternating Hemiplegia of Childhood (AHC). She also manifested episodes of psychomotor agitation and sleep disturbances with nocturnal awakenings. Ictal recording of one prolonged right hemiplegic event was associated with theta activity and epileptiform abnormalities on the right hemisphere. Flunarizine was added to her antiseizure treatment and was partially effective in reducing the frequency of her hemiplegic episodes. Niaprazine was also introduced and helped with her sleep disorder. The use of diazepam as needed was often effective to stop the prolonged paroxysmal events, both seizures and hemiplegic events.

Formal cognitive evaluations at 3 years and 5 years documented moderate-severe developmental delay (Griffith scale).

At last follow-up (age 5y) she was still experiencing multiple types of events on a weekly basis, including prolonged hemiplegic episodes, and shorter events with stiffness, eye deviation, oro-motor automatisms. She was on treatment with lacosamide, flunarizine and niaprazine. Lamotrigine was added recently leading to improved seizure control.

At age 5 years auxological parameters were: weight 17.6 Kg (43th p), height 114 cm (87th p), OFC 46.9 cm (<1st p, -2.6SD). Her speech was limited to vocalization and only disyllabic words. She had some motor impairment with unstable and mildly wide-based gait.

Brain and spine MRI showed multifocal cortical abnormalities with abnormal folding especially in the frontal regions and thickened cortical areas including insula and caudate nuclei, cerebellar vermis hypoplasia, mildly enlarged lateral ventricles, cervico-dorsal syringomyelia remaining stable over multiple evaluations (9m, 3 y and 5 y 3m).

Previous genetic investigations, including pre- and post-natal karyotype and array CGH, gave normal results. Trio exome sequencing revealed a homozygous variant of *TM2D3*: c.503G>A p.(Gly168Asp).
**Individual 4** is an almost 26 years old female who was born to non-consanguineous parents at 36 weeks of gestation age via spontaneous vaginal delivery with a history of multiple congenital anomalies, craniofacial dysmorphism, multiple hemangiomas, and a ventricular septal defect that spontaneously closed. The pregnancy was unremarkable with the exception of a maternal renal stone that required treatment. Prenatal ultrasounds were reportedly within normal limits. Birth weight was at the 25th centile, birth length at the 50th centile and birth head circumference at the 10th centile. She was first evaluated by Genetics at 2 months of age. At that time her weight was at the 25th centile, her length at the 30th centile and her head circumference at the 5th centile. Her craniofacial exam revealed narrow palpebral fissures, a beaked nose, micrognathia, some facial asymmetry with the left jawline less prominent than the right, and the left ear larger than the right ear. She was noted to have multiple hemangiomas including one on the left upper lip, one on the right neck, one in the left anterior shoulder, four on the back as well as one on the second digit of the right hand. There were additional flexion creases on all fingers. Gross and fine motor development were delayed and she did not walk until 21 months of age. There was no speech and language development and she continues to be non-verbal to this date. Now at 26 years of age she is hyperactive and walks with a broad based ataxic gait. She experiences hour long episodes of irritability and agitation associated with stereotyped movements. She had a CT-scan at 3 years showing a pineal cyst but no brain MRI was performed.

Extensive genetic testing throughout her life was non-informative including karyotype, single gene sequencing (MECP2, CDKL5/STK9, FOXG1, SLC9A6, TCF4, ARX, NLGN3, NLGN4, UBE3A). Chromosomal microarray revealed a paternally inherited 576kb duplication at 11p11.2.

Trio exome sequencing eventually revealed a heterozygous, maternally inherited variant in *TM2D3* c.677C>T; p.(Thr226Met) and a paternally inherited heterozygous multi-gene deletion of 15q26.3 that includes the *TM2D3* gene.
**Supplemental Note: Figures**

<figure><img src="image_17.png" /><figcaption>Figure S1</figcaption></figure>

**Figure S1: Brain and spine MRI of TM2D3 individuals.** Individual 1 at 3 years ½ on axial T1 (A) showing global hypotrophy predominant on frontal lobes. Individual 2 at 5 years on axial T1 (B) and axial T2 (C) showing small frontal lobes and abnormal gyration (white triangles). Individual 3 at 3 years on axial T1 (D), coronal T1 (E), brain and spine sagittal T2 (G-H) and 5 years 3 months on coronal T1 (F), showing a dysgyric cortical pattern with thickening and abnormal folding (white triangles), reduced volume of the frontal lobes, slightly increased and asymmetric ventricular size, mild hypoplasia of the inferior cerebellar vermis (black stars), and cervico-dorsal syringomyelia (thin arrows).
<figure><img src="image_18.png" /><figcaption>Figure S2</figcaption></figure>

**Figure S2:** Generation of SNB75 cells knockout for *TM2D3* gene by CRISPR-Cas9. *TM2D3* mRNA expression was confirmed in SNB75 WT or *TM2D3_KO* (n=5 biological replicates). β2-microglobulin (*B2M*) and *GAPDH* were used as housekeeping genes. Statistical analysis was performed using unpaired t tests, \*p\<0.05.
<figure><img src="image_19.png" /><figcaption>Figure S3</figcaption></figure>

**Figure S3: The Gly168Asp variant does not affect TM2D3 localization to the endoplasmic reticulum.**

A: SNB75 *TM2D3_KO* cells were transfected with plasmids carrying TM2D3_Gly168Asp/GFP (*p.TM2D3_Gly168Asp*), the short isoform of WT TM2D3 (*p.TM2D3_Short*) and a truncated variant lacking the predicted N-terminus signal peptide of 33 amino acids (*p.TM2D3_33-End*) and incubated with TMRM and ER-tracker® dyes. Representative images of 4 independent experiments. B: SNB75 WT cells were transfected with a plasmid carrying a sequence encoding the first 33 amino acids of TM2D3 fused to GFP (*p.N33_TM2D3-GFP*) and DsRed2-ER-5 plasmids to confirm the ER targeting. Images were obtained by confocal microscopy and fluorescence spectrums were obtained using ImageJ®. Representative images of 3 independent experiments. Scale bar: 20µm.
<figure><img src="image_20.png" /><figcaption>Figure S4</figcaption></figure>

**Figure S4: Expression analysis of endoplasmic reticulum stress genes in each individual cell line compared to their parents.** Fold change of ER stress transcripts *ATF4*, *HSPA5* and *DDIT3* in fibroblasts from parents (white, merged means of 3 biological replicates for each parent) or individuals carrying *TM2D3* variants (dark, n=3 biological replicates) after 24h Tunicamycin incubation (1µg/ml). \*p<0.05, \*\*p<0.01, \*\*\*p<0.001.
<figure><img src="image_21.png" /><figcaption>**Figure S5: Wild Type TM2D3 isoforms rescue the ER stress alterations in SNB75 TM2D3_KO cells.**

**Upper panel:** representative image (3 independent experiments) of the GRP78 (protein encoded by *HSPA5*) signals obtained with automated western blot for all conditions (p.TM2D3_WT= long isoform; p.TM2D3_Short=short isoform, p.TM2D3_Gly168Asp= p.(Gly168Asp) variant). **Lower panel:** Quantification of the GRP78 signals from 3 independent experiments. Statistical analysis was performed using unpaired t tests, \*p<0.05, \*\*p<0.01.</figcaption></figure>
<figure><img src="image_22.png" /></figure>

**Figure S6: Mitochondrial structure alterations in SNB75 and individuals' cells.** Mitochondrial length, cristae width and ER-Mitochondria distance are increased in SNB75 *TM2D3_KO* and in the individuals compared to their respective parents. For Individual II-1 of Family 3, we had no access to parents' fibroblasts. Mean of mitochondrial length, cristae width and ER-mitochondria distance were calculated from at least 50 different mitochondria or ER-mitochondria contacts per cell. Data are further expressed as mean±SEM of 3 independent cells for each condition (merged data for I-1 / I-2). Statistical analysis was performed using unpaired t tests, \*p<0.05, \*\*p<0.01, \*\*\*p<0.001, \*\*\*\*p<0.0001.
<figure><img src="image_23.png" /><figcaption>Figure S7</figcaption></figure>

**Figure S7: Mitochondrial fusion protein OPA1 is overexpressed in SNB75 TM2D3_KO cells:** Left: representative immunoblot showing the signals obtained for OPA1 and loading control protein α-Tubulin. Right: quantification of the normalized OPA1 signals from n=3 independent experiments. Statistical analysis was performed using unpaired t tests, *p<0.05.
<figure><img src="image_24.png" /><figcaption>Figure S8</figcaption></figure>

**Figure S8: Differential gene expression and functional enrichment analysis.** A: Volcano plot depicting the differentially expressed genes (DEGs). The x-axis represents the log2 fold change of gene expression, while the y-axis corresponds to the -log10 adjusted p-value. Genes significantly upregulated are in red and significantly downregulated are in blue. The vertical red lines indicate a log2 fold change threshold, and the horizontal red line represents the significance threshold for adjusted p-value. B: Gene Ontology (GO) enrichment analysis of DEGs. The x-axis represents the percentage of genes in each term relative to the total number of DEGs. The y-axis shows the significantly enriched GO terms. The color gradient from blue to white indicates the p-value, with blue representing more significant terms. Circle size denotes the intersection size, i.e., the number of genes associated with each GO term.
## Supplemental Note: Tables

<caption>Table S1</caption>

### A.

<table><thead><tr><th></th><th colspan="2">Controls</th><th>II-1 Family 1</th><th>II-1 Family 2</th></tr></thead><tbody><tr><td>CS</td><td>529±116</td><td>(350 - 840)</td><td>537</td><td>557</td></tr><tr><td>I/CS</td><td>0.50±0.07</td><td>(0.36 - 0.62)</td><td>0,52</td><td>0,43</td></tr><tr><td>II/CS</td><td>0.17±0.03</td><td>(0.11 - 0.25)</td><td>0,21</td><td>0,21</td></tr><tr><td>III/CS</td><td>1.15±0.28</td><td>(0.64 - 1.70)</td><td>1,47</td><td>0,95</td></tr><tr><td>IV/CS</td><td>1.09±0.19</td><td>(0.72 - 1.63)</td><td>1,05</td><td>0,90</td></tr><tr><td>V/CS</td><td>0.40±0.09</td><td>(0.21 - 0.55)</td><td>0,55</td><td>0,43</td></tr></tbody></table>

### B.

<table><thead><tr><th></th><th colspan="2">Controls</th><th>II-1 Family 1</th><th>II-1 Family 2</th></tr></thead><tbody><tr><td>St3 MP</td><td>19.71±4.17</td><td>(11.38-28.06)</td><td>14,71</td><td>16,33</td></tr><tr><td>St3 MPG</td><td>20.36±4.26</td><td>(11.84-28.89)</td><td>14,51</td><td>16,58</td></tr><tr><td>St3 MPGS</td><td>26.19±5.42</td><td>(15.34-37.05)</td><td>17,96</td><td>22,59</td></tr><tr><td>St3 SR</td><td>15.96±2.96</td><td>(10.05-21.87)</td><td>12,79</td><td>15,77</td></tr><tr><td>COX</td><td>32.77±7.90</td><td>(16.96-48.57)</td><td>32,21</td><td>35,32</td></tr></tbody></table>

**Table S1: Exploration of mitochondrial activity in individuals' cells.** A: Activities of Citrate synthase, respiratory chain complexes and F1-ATPase normalized to citrate synthase. Activities were measured on mitochondrial enriched fraction from patients and controls cells. Citrate synthase activity is expressed in nmol/min/mg proteins. Patients: analyses were performed on two biological replicates, each in duplicate. Controls: mean±S.D. of one hundred independent fibroblast control samples, used to define the reference range (bracket). B: Maximal phosphorylating respiration. Phosphorylating respirations (st3: states 3) measured on permeabilized fibroblasts, successively respiring with either CI substrates (malate and pyruvate + ADP: St3 MP or malate, pyruvate and glutamate + ADP: St3MPG), CI+ CII substrates (+ succinate, St3 MPGS), CII-linked respiration (succinate + rotenone, St3 SR). COX: maximal complex IV oxidation rate, measured using the TMPD+ascorbate couple. Oxygen consumptions are expressed in nmol.min-1.mg proteins-1. Patients: analyses were performed on three biological replicates, each in duplicate. Controls: mean±S.D. of one hundred independent fibroblast control samples, used to define the reference range (bracket).
**<u>Supplemental Note: Methods</u>**

**Exome sequencing and analysis**

Single-nucleotide *TM2D3* variants were all identified by exome sequencing on proband in family 1 and on probands and their parents for family 2, 3 and 4. Genomic DNA was extracted from whole blood of the affected individual and underwent exome sequencing in a clinical or research local setting using Illumina short-read sequencing. Reads were aligned to the human reference genome sequence (National Center for Biotechnology Information [NCBI] build 37.3, hg19). Bioinformatic pipelines annotated, filtered, and prioritized high-quality variants that differed from the reference sequence into a small number of clinically relevant variants as described previously. Variant filtration was performed utilizing gnomAD frequency data and bioinformatic prediction algorithms. Initial analysis of neurodevelopmental disorder (NDD)-associated genes yielded inconclusive results. Subsequently, *TM2D3* was identified as a candidate gene in individual 1, based on the detection of a stopgain variant in conjunction with a rare, predicted pathogenic missense variant. Parental Sanger sequencing confirmed compound heterozygosity of these variants. GeneMatcher facilitated connections with other research teams that had identified similar genotypes. In all cases, the *TM2D3* variants identified in probands were searched for in their parents by targeted Sanger sequencing and in the sister of individual 2. The variants were submitted to the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/). *TM2D3* sequence analysis for mutation intolerance and predictive 3D-folding were performed with MetaDome² and Alphafold³ respectively.

**RT PCR cDNA analysis**

To assess the predicted impact of the c.503G>A variant on splicing, blood samples from PAXgene Blood RNA tubes were collected from individual 1 and his parents. Total RNA was isolated with the NucleoSpin RNA Plus Kit (Macherey-Nagel) according to the manufacturer's instructions. The concentration of total RNA was measured on a NanoDrop spectrophotometer (NanoDrop Technologies). Reverse transcription was performed with the ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs). PCRs were performed on cDNA with the HotStarTaq DNA Polymerase (Qiagen) according to the PCR amplicons (PCR conditions are available upon request). PCR products were analyzed on 1% agarose gel electrophoresis and Sanger sequenced.

**Generation of TM2D3-KO cells via CRISPR/Cas9**

Plasmids pX330 encoding for Cas9 and 3 guide-RNAs targeting TM2D3 exon 1 [(5'-3') target site sequence: G1: GGAGGGGTGCTCCCGCTGAGGGG, G2: CAGTTCTGCATTCTGTCGGGCGG, G3: TTCTGCATTCTGTCGGGCGGTGG)], were co-transfected with a puromycin-resistant gene flanked by homology arms upstream and downstream of the target site. Transfected cells
were selected with 1 µg/mL puromycin at 72 h after initial transfection. PCR with primers upstream and downstream of the target site confirmed the absence of the TM2D3 PCR product in KO cells [5'-3' (E1-F1: AAAGGACGAAACACCGGAGGGGTGCTCCCGCTGAGGTTTTAGAGCTAGAA, E1-R1: TTCTAGCTCTAAAACCTCAGCGGGAGCACCCCTCCGGTGTTTCGTCCTTT, E1-F2: AAAGGACGAAACACCCAGTTCTGCATTCTGTCGGGGTTTTAGAGCTAGAA, E1-R2: TTCTAGCTCTAAAACCCCGACAGAATGCAGAACTGGGTGTTTCGTCCTTT, E1-F3: AAAGGACGAAACACCTTCTGCATTCTGTCGGGCGGGTTTTAGAGCTAGAA, E1-R3: TTCTAGCTCTAAAACCCGCCCGACAGAATGCAGAAGGTGTTTCGTCCTTT)]. Secondary screen by PCR using two set of primers [5'-3' (F1: CCATCTGTTACCTGTGTTGATCAAG, R1: GAGGACAGGACACCGTCATG and, F2: AACTCCACCAGCTGCATGAC, R2: TAGACCACTTATAGCCTCCAGTCC) was carried out in 24 colonies to identify clones with different levels of TM2D3 expression. For further experiments, we selected the clone with the lowest expression (0.6%, see Figure S2).

## Cell culture

SNB75 glioblastoma cells (WT and TM2D3_KO) were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) (Corning®) and 1% Glutamine (Dutscher®). Fibroblasts were obtained from skin biopsies of individuals carrying the G168D TM2D3 variants with informed consent and cultured in DMEM/Nutrient Mixture (F12) supplemented with 10% Fetal Bovine Serum (FBS) (Corning®), 33% Amniomax (Gibco®), 1% Uridine (Dutscher®) and 1% Pyruvate (Dutscher®). All cultures were maintained at 37°C in a humidified incubator with 5% CO₂ and experiments were conducted on cells with similar passage numbers, ranging from 6 to 25, to avoid possible senescence effects. Absence of mycoplasma contamination was routinely controlled. For biochemical experiments, cells were washed with PBS, trypsinized, centrifugated (800 rpm, 5 min), washed in PBS, and pellets were either immediately used for oxygraph analyses or frozen in liquid nitrogen and stored at -80°C until use for enzymatic measurements.

## Mitochondrial function exploration

OXPHOS enzymes (complex I to IV of the mitochondrial respiratory chain and F1-ATPase) and citrate synthase activities were assessed at 37°C with a UVmc2 spectrophotometer (SAFAS, Monaco) on mitochondrial enriched fractions from the fibroblasts according to standard routine clinical protocols⁴. Mitochondrial oxygen consumption measurements were performed at 37°C and atmospheric pressure using a high-resolution oxygraph (O2K, Oroboros Instrument, Innsbruck, Austria).

Respiration rates on permeabilized cells (digitonin, 15 µg/106 cells) were measured in respiratory buffer RB (10mM KH2PO4, 300mM mannitol, 10mM KCl, 5mM MgCl2, 0.5mM EGTA and 1 mg/ml serum albumin bovine, pH7.2) using substrates of Cl, Cl+ClI and ClI as followed: first, state 2 (non-phosphorylating) respiration was measured after adding 2.5mM
pyruvate and 5mM malate. Then, the CI-linked maximal phosphorylating respiration (state 3) was stimulated by saturating ADP concentration (1.5mM) and 3mM NAD+, added to avoid TCA limitation by NAD+ availability. Succinate (10mM) was then added to measure the combined CI and CII-linked respiration with convergent CI+II electron flow into the Q-junction corresponding to the maximal stimulated phosphorylating respiration (OXPHOS capacity). Rotenone (5µM) was used to inhibit CI activity and thus to obtain the maximal CII-linked respiration. Thirdly, oligomycin (FOF1-ATP synthase inhibitor, 4µg/ml) and FCCP (carbonyl cyanide p-trifluoromethoxyphenylhydrazone, a mitochondrial uncoupler, 1µM) were sequentially added to ensure that the cells were fully permeabilized. Antimycin A (2µg/ml) was then used to inhibit complex III and check for the non-mitochondrial oxidation. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD, 0.3 mM) reduced by ascorbate (4 mM) were then added as artificial donor for cytochrome c oxidase, lastly inhibited using KCN (1 mM) and sodium azide (2 mM). Cells protein content within the oxygraphic chambers was determined with the bicinchoninic assay kit (Uptima, Interchim, Montluçon, France) using bovine serum albumin as standard.

## Plasmids construction, cell transfection and confocal microscopy

To determine the cellular localization of the wild-type and mutated TM2D3 proteins, we designed several GFP-tagged plasmids. First, we cloned the canonical coding sequence of TM2D3 (from the commercial plasmid NM_078474, Origene) into a plasmid with GFP-tag (pEGFP-N1, Addgene). Second, we used this TM2D3-WT-GFP plasmid to generate a Gly168Asp variant via directed mutagenesis using the primers [5'-3' (Gly168Asp-F2: TGCTTGGATAACCGTACTTTTCCC and Gly168Asp-R2: ACGGTTATCCAAGCAGTGGAC)] with the In-fusion cloning kit (Takara Biotech). We also cloned the first 33 amino acids of canonical TM2D3 predicted to be a signal peptide (Interpro prediction, https://www.ebi.ac.uk/interpro/) fused to GFP (N33-TM2D3-GFP) as well as a truncated variant of TM2D3 lacking these 33 first amino acids (p.TM2D3_33-End). SNB75 TM2D3_KO cells were seeded in 4-well Ibidi®chamber coverslips (30 000 cells per chamber) the day before the experiment. On the day of the experiment, cells were washed once with Phosphate Buffered Saline (PBS) and put in 200µl fresh complete medium. 1µg of plasmids containing either TM2D3_WT or TM2D3_Gly168Asp sequences were put in Eppendorf tubes containing FBS-free medium, then an equal volume of TransIT-X2® Dynamic Delivery System (Mirus) was added. After 40 min of incubation, 200µl of plasmids were added to wells and let for 6h at 37°C in a humidified incubator with 5% CO₂. Then, transfection medium was removed, and fresh complete medium was added for a total time of 24h. Then, SNB75 TM2D3_KO cells were stained with the mitochondrial probe TMRM (1/4000) and with the ER probe ER-Tracker™ Blue-White DPX (1µM) for 15 minutes. Medium was removed, and fresh medium was added for confocal microscopy. All images were collected with a Leica TCS SP8 AOBS confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) equipped with a HC PL APO CS2 40X/NA 1.10 water objective and gateable hybrid detectors (GaAsP).
Images were acquired in the format 1024x1024 pixels, bit depth of 8, and a scanspeed of 400Hz. Z-series optical sections were collected with a step-size of 1 µm using a Super Z Galvo Type H stage and were displayed as maximum z-projections using the LAS X software.

Targeting of TM2D3 to the ER was further confirmed by co-transfection of the p.N33(TM2D3-GFP) plasmid with an ER-tag plasmid (DsRed2-ER-5, Addgene, #55836) in wild-type SNB75 cells.

## ER stress induction

SNB75 cells and primary skin fibroblasts were seeded on 6-well plates (300 000 cells per well) the day prior to the experiment. On the day of the experiment, cells were washed once with PBS. To induce ER stress, fresh medium containing 1µg/ml of Tunicamycin (Sigma Aldrich) was then added to the cells that were further maintained at 37°C in a humidified incubator with 5% CO2 for 24h. Cells were then collected using 10%v/v Trypsin, washed once using PBS and frozen as cell pellets for subsequent experiments.

## Quantitative PCR

Total RNA was extracted from SNB75 cells and individuals' fibroblasts using the RNeasy™ Mini Kit (Qiagen) according to manufacturer's instructions. RNA concentrations and purity were assessed with a NanoDrop™ 8000 Spectrophotometer (Thermo Scientific™). RNA was then retrotranscribed into cDNA using the SuperScript™ Reverse Transcriptase (Thermo Scientific™). qPCR was performed using SYBR Green (Thermo Scientific™) with specific primers targeting *ATF4* ([5'-3'] F: GTTCTCCAGCGACAAGGCTA, R: ATCCTGCTTGCTGTTGTTGG), *HSPA5* ([5'-3'] F: TGTTCAACCAATATCAGCAAACTC, R: TTCTGCTGTATCCTCTTCACCAGT), and *DDIT3* ([5'-3'] F: AGAACCAGGAAACGGAAACAGA, R: TCTCCTTCATGCGCTGCTTT). Analysis was performed on a CFX Connect™ Real-Time Detection System (BioRad®) and gene expression was calculated with the 2^ΔΔCt method using β2-microglobulin ([5'-3'] F: CAGCTCTAACATGATAACC, R: CCTGTAGGATTCTTCTTTC) as the reference gene.

## Western blot

**GRP78:** SNB75 WT or *TM2D3_KO* cells, transfected with the plasmids p(TM2D3_WT), p(TM2D3_Gly168Asp), and p(TM2D3_Short, were harvested after treatment or not with Tunicamycin (1µg/ml for 24h). Cell pellets were lysed in RIPA buffer (1X) supplemented with protease inhibitors (cOmplete Protease Inhibitor Tablets, Roche®) for 1.5 hours at 4°C and then centrifuged for 30 minutes at 21,000g (4°C). The protein concentration in supernatants was then quantified using the Pierce BCA Kit (ThermoFisher™). GRP78 expression was determined by Simple Western, a microfluidic-based technique for protein separation and detection. Using the Jess system (ProteinSimple, BioTechné®), samples (0,5mg/ml) are separated by capillary electrophoresis, transferred onto a matrix, and probed with GRP78 antibody (1:400, C50B12, Cell Signaling Technology). Detection is achieved through anti-Rabbit-HRP antibody (042-206, BioTechné®) and Luminol/Peroxide exposure. GRP78
expression was normalized on total protein detected in all capillaries using the Protein Normalization Module (DM-PN02, BioTechne®). For OPA1 expression, 40 µg of protein samples were loaded on 8% acrylamide gels and transferred using the iBlot transfer system (ThermoFisher) ™ onto nitrocellulose membranes. OPA1 antibody (MAB9506-100, 1:1000, R&D Systems) and α-Tubulin (ab7291, 1:5000, Abcam) were incubated overnight at 4 °C. Secondary antibodies (anti-Rabbit Abcam ab186696, 1:10000 and anti-Mouse Abcam ab186695, 1:10000) were then incubated for 1 h at room temperature. Proteins were detected with fluorescence at 680 and 790nm using the LI-COR imaging system (LICORbio®). Quantification by densitometry was performed with ImageJ software (NIH).

## Electronic microscopy

SNB75 cells and individuals' fibroblasts were seeded in 6-well culture plates and fixed for 16 hours at 4°C with 2.5% glutaraldehyde (LFG Distribution, Lyon, France) in 0.1M phosphate buffer pH7.4. Samples were then rinsed in phosphate buffer 0.1M pH7.4. After a rapid rinse in milliQ water, the samples were postfixed with 1% osmium tetroxide / 1% potassium ferricyanide in milliQ water for 1 hour at room temperature. Samples were then rinsed with milliQ water and dehydrated in a graded series of ethanol, and then embedded in EMbed 812 epoxy resin (LFG Distribution) at 37°C for 24 hours, 45°C for 24 hours and 60°C for 72h. Thin sections (60nm-thickness) were cut with an EM-UC7 ultramicrotome (Leica, Nanterre, France), contrasted on a droplet of 3% uranyl acetate for 15 minutes, and observed with a Jeol JEM 1400 microscope (Jeol, Croissy sur Seine, France) operating at 120 KeV.

## RNA sequencing

RNA from fibroblasts was extracted using the Monarch® RNA Purification Kit (New England Biolabs), according to the supplier's recommendations. The extracted RNA was qualified by determining the degradation index (RIN) on a Bionanalyzer (Agilent), quantified (Qubit RNA HS Assay Kit, Thermofischer), according to the supplier's recommendations, and stored at -80°C.

After mRNA enrichment by PolyA capture ("NEBNext® Poly(A) mRNA Magnetic Isolation Module, New England Biolabs), the cDNA libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep kit (New England Biolabs) according to the supplier's recommendations. The cDNA libraries were qualified by capillary electrophoresis on a bioanalyzer (Agilent) and quantified at Qubit using the dsDNA HS Assay kit (Thermofischer). Libraries were paired-end sequenced on the NextSeq550 platform (Illumina®) with NextSeq 500/550 High Output Kit v2.5 (150 cycles). Sequencing data were processed through a homemade bioinformatics pipeline. Raw data were converted in fastq using the module GenerateFastq (Illumina®), and a quality check was performed by Fastqc (v 0.11.9).
Sequencing reads were mapped to the human genome GRCh38 from the Genome Reference Consortium using STAR (Spliced Transcripts Alignment to a Reference) v2.7.10a⁴. Quantification of reads was performed by featureCounts from Subread⁵ using the GENCODE annotation file (gencode.v38 annotation.gtf). Differential gene expression was analysed using DESeq⁶. The functional enrichment analysis was performed using Groovy and PANTHER (http://pantherdb.org/). The graphs were created with R (version 4.2.1)⁸. The complete raw dataset is available in the BioProject Database (BioProject ID: PRJNA1185456).

**Statistical analysis**

Data were analysed using GraphPad Prism 8.0 for Windows. Normality of the data distribution was assessed using a Shapiro-Wilk test. A non-parametric test (Mann-Whitney U test for unpaired data) was used to compare two groups where data were not normally distributed. A parametric test (unpaired t test for unpaired data) was used to compare two groups where data were normally distributed. A value of p<0.05 was considered statistically significant. No adjustment for multiple comparisons was made.
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