Bi-allelic loss-of-function variants in TMEM147 cause moderate to profound intellectual disability with facial dysmorphism and pseudo-Pelger-Huët anomaly

Abstract

The transmembrane protein TMEM147 has a dual function: first at the nuclear envelope, where it anchors lamin B receptor (LBR) to the inner membrane, and second at the endoplasmic reticulum (ER), where it facilitates the translation of nascent polypeptides within the ribosome-bound TMCO1 translocon complex. Through international data sharing, we identified 23 individuals from 15 unrelated families with bi-allelic TMEM147 loss-of-function variants, including splice-site, nonsense, frameshift, and missense variants. These affected children displayed congruent clinical features including coarse facies, developmental delay, intellectual disability, and behavioral problems. In silico structural analyses predicted disruptive consequences of the identified amino acid substitutions on translocon complex assembly and/or function, and in vitro analyses documented accelerated protein degradation via the autophagy-lysosomal-mediated pathway. Furthermore, TMEM147-deficient cells showed CKAP4 (CLIMP-63) and RTN4 (NOGO) upregulation with a concomitant reorientation of the ER, which was also witnessed in primary fibroblast cell culture. LBR mislocalization and nuclear segmentation was observed in primary fibroblast cells. Abnormal nuclear segmentation and chromatin compaction were also observed in approximately 20% of neutrophils, indicating the presence of a pseudo-Pelger-Huët anomaly. Finally, co-expression analysis revealed significant correlation with neurodevelopmental genes in the brain, further supporting a role of TMEM147 in neurodevelopment. Our findings provide clinical, genetic, and functional evidence that bi-allelic loss-of-function variants in TMEM147 cause syndromic intellectual disability due to ER-translocon and nuclear organization dysfunction.

Keywords: DNA methylation; LBR; Pelger-Huët anomaly; TMEM147; facial dysmorphism; intellectual disability; neurodevelopmental disorder; nuclear envelope instability; transcriptomics; translocon dysfunction.

Graphical abstract

Figure 1

Individuals with TMEM147 germline variants identified in the cohort (A) Representation of TMEM147 (purple) with its seven helices. Metadome constraint plot and distribution of exomic regions are reported above the TMEM147 model. Families with disease-causing variants are reported below. Homozygous variants are indicated in bold. Nonsense and frameshift variants are indicated in red, splice-site variant in blue, and missense variant in black. (B) Family pedigrees and segregation analysis of the identified variants. “A” represents the wild-type allele and “a” the mutated one.

Figure 2

Photos of the affected individuals included in the cohort and brain MRI results (A) Affected individuals showed consistent coarse facial features. A merged image was obtained by the Facer tool. (B) Brain MRI of individuals with TMEM147 disease-causing variants. Thin corpus callosum is present in all individuals whose brain MRIs were available. Subcortical atrophy with ventricle enlargement is observed in all individuals except i22 (F15-V-1) and i9 (F6-V-6) and was more pronounced in i10 (F6-V-7) at 10 months of age, associated with a cortical atrophy. According to age, myelination remains poor on MRI performed at 5 years of age (i5 – F4-IV-9, i6 – F5-IV-2, and i9 – F6-V-6), particularly in the temporal white matter with a hypersignal flair of the periventricular white matter in two individuals.

Figure 3

Structural analysis of the p.Gly7Arg, p.Ile133Asn, and p.Arg166Trp variants (A) Cartoon representation of the ER translocon structure (PDB: 6W6L). The five proteins involved in the transmembrane channel are colored: Nicalin (yellow), Sec61 (green), TMCO1 (cyan), CCDC47 (purple), and TMEM147 (pink). Ribosomal proteins and RNA are presented in gray. Membrane position is indicated with the lumen. Nascent protein sequence is shown as orange spheres inside the ribosome structure. (B) Structural representation of TMEM147 and its main partners. For better visualization, ribosomal proteins and RNAs have been masked. Three dotted squares indicate the magnified regions used to model the variant effects. Squares 1, 2, and 3 indicate the enlarged regions used to model the p.Ile133Asn, p.Gly7Arg, and p.Arg166Trp variants, respectively. (C) Cartoon representation of TMEM147 presenting the three enlarged regions. In the larger dotted inserts, the wild-type situation for each amino acid is displayed as stick. 1, Ile133; 2, Gly7; and 3, Arg166. In the adjacent insert, the mutated amino acid is modeled as a comparison. Only the side chains are represented in purple, leaving the α-carbon as a pink ribbon representation. Hydrogens have been masked for better visualization. (D) (1, left) Surface rendering of the Ile133 area. The α-helix faces the carboxy-terminal region of Nicalin. (1, right) Surface rendering of the Ile133Asn variant as a model. From the 11 possible rotamers for asparagine in this context, the one causing the minimum of constraints was selected. (2, left) Surface rendering of the Gly7 area. The molecule was rotated by 90° and viewed from above. (2, right) Surface rendering of the Gly7Arg variant as a model. From the 24 possible rotamers for arginine in this context, all of them are causing mild to severe steric clashes with TMEM147 itself or the surrounding polypeptidic chains. The rotamer causing the lowest degree of perturbation was arbitrarily selected. (3, left) Surface rendering of the Arg166 area. (2, right) Surface rendering of the p.Arg166Trp variant as a model. From the seven possible rotamers for tryptophan in this context, all of them are causing mild steric clashes with TMEM147. The rotamer causing the lowest degree of perturbation was arbitrarily selected. Dotted shapes indicate the positions of the amino acids in the chain.

Figure 4

Biochemical characterization of the TMEM147 mutant proteins and immunostainings in fibroblasts (A) Accelerated degradation of the TMEM147^Arg7Gly (R7G), TMEM147^Ile133Asn (I133N), and TMEM147^Arg166Trp (R166W) proteins. Immunoblot analysis shows WT and variant V5-tagged TMEM147 protein levels in transfected COS-1 cells, basally and after CHX (10 μg/mL) or bafilomycin (200 nM) treatment. GAPDH was used as loading control. Representative blots (below) and mean ± SD densitometry values (above) of three independent experiments are shown. Asterisks indicate statistically significant differences compared with WT TMEM147 (^∗∗∗∗p ≤ 0.0001; ^∗∗∗p ≤ 0.001; ^∗∗p ≤ 0.05; two-way ANOVA followed by Tukey’s multiple comparison test). (B) Subcellular localization of transiently expressed V5-tagged WT or mutant TMEM147 proteins in COS-1 cells under steady-state conditions revealed by confocal microscopy analysis. Cells were stained with the anti-V5 monoclonal antibody (red). Co-localization analysis was performed using the endoplasmic reticulum marker calnexin (green). Merged images with nuclei (Hoechst 33342 staining, blue) are displayed on the right. Scale bar, 10 μm. (C) Quantification of mean fluorescence signals ± SEM detected in (D). Three technical replicates were performed per cell line. A total of 150 measurements per cell line were performed. Asterisks indicate statistically significant differences compared with cell lines from healthy individuals: control 1, control 2, and control 3 (^∗∗∗∗p ≤ 0.0001; ^∗∗∗p ≤ 0.001; ^∗∗p ≤ 0.01; ^∗p ≤ 0.05; one-sample Wilcoxon test, based on the average of the three control samples). (D and E) Localization analysis of CKAP4 (CLIMP-63) (D) and RTN4 (E) in fibroblasts from i1, i22, and i23 and healthy control individuals (only control 1 is shown in the figure). Scale bar, 20 μm.

Figure 5

LBR localization, nuclear morphology in fibroblasts, and Pelger-Huët-like anomaly in granulocytes (A) Localization analysis of LBR and actin in fibroblasts from i1, i22, and i23 and healthy control individuals (only control 1 is shown in the figure). Scale bar, 20 μm. The inset in control 1 line shows a higher magnification of the nucleus indicated by the dashed square. Actin staining shows that TMEM147 does not affect gross cell morphology. (B) Nuclear segmentation is observed in fibroblast cell nuclei from affected individuals. Fibroblasts were stained with the May Grunwald-Giemsa (MGG) method. (C) Quantification of the nuclear segmentation observed in fibroblast cell lines. (D) Chromatin clumping, hyposegmentation, bilobed nuclei, carioschizes, and drumsticks (red arrows) observed in neutrophils of i1.