Deficiencies in vesicular transport mediated by TRAPPC4 are associated with severe syndromic intellectual disability

Abstract

The conserved transport protein particle (TRAPP) complexes regulate key trafficking events and are required for autophagy. TRAPPC4, like its yeast Trs23 orthologue, is a core component of the TRAPP complexes and one of the essential subunits for guanine nucleotide exchange factor activity for Rab1 GTPase. Pathogenic variants in specific TRAPP subunits are associated with neurological disorders. We undertook exome sequencing in three unrelated families of Caucasian, Turkish and French-Canadian ethnicities with seven affected children that showed features of early-onset seizures, developmental delay, microcephaly, sensorineural deafness, spastic quadriparesis and progressive cortical and cerebellar atrophy in an effort to determine the genetic aetiology underlying neurodevelopmental disorders. All seven affected subjects shared the same identical rare, homozygous, potentially pathogenic variant in a non-canonical, well-conserved splice site within TRAPPC4 (hg19:chr11:g.118890966A>G; TRAPPC4: NM_016146.5; c.454+3A>G). Single nucleotide polymorphism array analysis revealed there was no haplotype shared between the tested Turkish and Caucasian families suggestive of a variant hotspot region rather than a founder effect. In silico analysis predicted the variant to cause aberrant splicing. Consistent with this, experimental evidence showed both a reduction in full-length transcript levels and an increase in levels of a shorter transcript missing exon 3, suggestive of an incompletely penetrant splice defect. TRAPPC4 protein levels were significantly reduced whilst levels of other TRAPP complex subunits remained unaffected. Native polyacrylamide gel electrophoresis and size exclusion chromatography demonstrated a defect in TRAPP complex assembly and/or stability. Intracellular trafficking through the Golgi using the marker protein VSVG-GFP-ts045 demonstrated significantly delayed entry into and exit from the Golgi in fibroblasts derived from one of the affected subjects. Lentiviral expression of wild-type TRAPPC4 in these fibroblasts restored trafficking, suggesting that the trafficking defect was due to reduced TRAPPC4 levels. Consistent with the recent association of the TRAPP complex with autophagy, we found that the fibroblasts had a basal autophagy defect and a delay in autophagic flux, possibly due to unsealed autophagosomes. These results were validated using a yeast trs23 temperature sensitive variant that exhibits constitutive and stress-induced autophagic defects at permissive temperature and a secretory defect at restrictive temperature. In summary we provide strong evidence for pathogenicity of this variant in a member of the core TRAPP subunit, TRAPPC4 that associates with vesicular trafficking and autophagy defects. This is the first report of a TRAPPC4 variant, and our findings add to the growing number of TRAPP-associated neurological disorders.

Keywords: autophagy; intellectual disability; molecular genetics; vesicular transport; whole-exome sequencing.

Figure 1

Clinical and brain imaging features of affected subjects with TRAPPC4-related neurological disorder. (A) Frontal clinical images of affected subjects, noting the common features of microcephaly, facial dysmorphism, bitemporal narrowing and long philtrum. (B) T₂-weighted axial images show that between 6 months and 2 years of age Subject 1:II-1 had progressive generalized severe cerebral atrophy, particularly affecting white matter, with relative sparing of the basal ganglia. (C) T₂-weighted images showed that between 3 weeks and 6 years Subject 1:II-3 had progressive large extra-axial spaces and enlarged ventricles, plagiocephaly involving the occipital parietal region, with a thin corpus callosum and wide Sylvian fissures. (D) T₂ FLAIR SE axial image of Subject 2:II-5 at 6 months of age showing severe cerebral atrophy, and relatively milder cerebellar atrophy. There was preservation of deep grey matter structures. (E) T₂-weighted images of Subject 3:II-2 in the first year of life showed cerebral atrophy. Full clinical features are described in Supplementary Table 1.

Figure 2

Pedigrees of families and genetic findings in TRAPPC4-deficient families. (A) Pedigrees of Families 1–3 all with a recessive inherited c.454+3A>G variant in TRAPPC4. Confirmatory Sanger sequencing in the three families shows a homozygous c.454+3A>G TRAPPC4 variant in the affected subjects, a heterozygous variant in each parent and homozygous wild-type or heterozygous configuration in unaffected tested siblings. Asterisk indicates exome sequencing. (B) Multiple sequence alignment surrounding the variant was performed using the Multiz Alignment of 100 vertebrates in the UCSC browser shows that the c.454+3A>G TRAPPC4 variant is a highly conserved intronic residue (surrounded by red box). (C) The structure of TRAPPC4 (NM_016146.5) with the reference sequence of exon-intron junctions shown, and the c.454+3A>G variant indicated in red. (D) The heat map showing the allele count in gnomAD for the variants in splicing donor (SD) and splicing acceptor (SA) sites in TRAPPC4. Yellow colour shows higher frequency and blue colour shows the lower frequency allele counts. (E) RT-PCR analysis demonstrated a splicing defect leading to incomplete exon skipping and formation of an additional PCR product in an affected subject (Subject1:II-1, patient marked with a red arrow) that was not present in the affected subject’s parents (Subjects 1:I-1 and 1:I-2) or four controls (C1–C4). (F) Sanger sequencing of the RT-PCR fragments in E confirmed the sequence of the 362-bp product contained exons 2, 3 and 4 in controls, and in the affected subject (Subject 1:II-1), whilst the lower band only present in the affected subject confirmed skipping of exon 3. (G) Quantitative real-time PCR in control, parental and subject fibroblasts showed a significant decrease in the regular splice product of TRAPPC4 in Subject 1:II-1 fibroblasts and one of the parents (Subject 1:I-2) compared to controls. Additionally, there is a significant increase in levels of the aberrant transcript in fibroblasts from Subject 1:II-1 compared to controls. Statistical significance was determined using one-way ANOVA with Bonferroni correction for multiple comparisons. Data show mean, upper and lower limits of n = 3 independent biological collections. *P < 0.05; ***P < 0.001 versus C1.

Figure 3

Decreased protein levels and impaired TRAPP complex assembly in fibroblasts from an affected subject with TRAPPC4 c.454+3A>G homozygous variant. (A) Western blot of TRAPPC4 and GAPDH levels in four paediatric controls (C1–C4), as well as fibroblasts from an affected subject with c.454+3A>G homozygous variant (Subject 1:II-1) and parents (heterozygous for the same variant; Subjects1:I-1 and 1:I-2) in triplicate. (B) Quantification of the western blots in A demonstrate that there was a significant decrease in endogenous TRAPPC4 protein levels normalized to GAPDH in fibroblasts from an affected subject (Subject 1:II-1) compared to both parental fibroblasts (Subjects 1:I-1 and 1:I-2) and paediatric control fibroblasts. (C) Western blot of TRAPPC2 and GAPDH in control fibroblasts and an affected subject (Subject 1:II-1) and (D) quantification of western blots in C demonstrate no difference in protein levels of TRAPPC2 in Subject 1:II-1. (E) Western blot of TRAPPC12 and GAPDH in a control fibroblast and an affected subject (Subject 1:II-1) and (F) quantification of western blots in E demonstrate no difference in protein levels of TRAPPC12 in Subject 1:II-1. (G) Native PAGE electrophoresis revealed decreased levels of TRAPPC4-containing complexes in fibroblasts from Subject 1:II-1 relative to parental fibroblasts (Subjects 1:I-1 and 1:I-2) and paediatric control fibroblasts (C1–C4). Molecular size standards are indicated to the left. (H) Size exclusion chromatography of fibroblasts from Subject 1:II-1 and control fibroblasts. The TRAPP proteins TRAPPC8, TRAPPC12 and TRAPPC2L are shifting to a smaller molecular size in fibroblasts from the affected subject compared to control fibroblasts. The numbers at the top represent the fraction off the column, with smaller numbers indicating larger molecular size. Data in B, D and F are box-and-whisker plots showing median, interquartile interval, minimum and maximum of n > 3 per measurement from at least three biological collections of an affected subject and at least two independent experiments. Statistical significance was determined using one-way ANOVA with Bonferroni correction for multiple comparisons. *P < 0.05; **P < 0.01; ***P < 0.001. Molecular weight standards are indicated on the right (kDa).

Figure 4

Impaired Golgi trafficking in fibroblasts from an affected subject is rescued by lentiviral transduction of wild-type TRAPPC4. Control fibroblasts and fibroblasts derived from Subject 1:II-1 were either untransduced (Untx) or stably transduced with wild-type TRAPPC4 lentivirus (TRAPP). Cells were then transfected with a temperature-sensitive vesicular stomatitis virus glycoprotein VSVG–GFP ts045, then incubated at the non-permissive temperature of 40°C overnight. Cells were then shifted to the permissive temperature of 32°C for the time intervals indicated, fixed with PFA then immunostained with the Golgi marker GM130 and counterstained with the nuclear marker DAPI. VSVG-GFP ts045 co-localization with GM130 was determined for a minimum of 60 cells per time point and treatment. (A) There was no difference in trafficking in naïve control cells or those transduced with TRAPPC4 lentivirus. However, in fibroblasts from the affected subject there was a significant delay in VSVG-GFP ts045 entering the Golgi, and a significant delay in exiting the Golgi compared to control cells. Golgi trafficking in fibroblasts from the affected subject was restored back to levels of control upon transduction with wild-type TRAPPC4 lentivirus. (B) Representative confocal images of intracellular trafficking (blue, DAPI; green, VSVG-GFP ts045; red, GM130). More representative figures can be found in Supplementary Figs 1 and 2. Data are average ± SD, n > 60 cells per treatment and time point. Significance was measured by a two-way ANOVA with Sidak multiple comparisons correction. ***P < 0.001, ****P < 0.0001. Scale bar = 20 µm.

Figure 5

Autophagy is defective in fibroblasts from an affected subject. (A) Control fibroblasts and fibroblasts derived from a subject with the homozygous c.454+3A>G variant in TRAPPC4 (Subject 1:II-1) were either unstarved (0) or transferred into starvation medium (EBBS) for 2 h without (2) or with (2B) bafilomycin A1. The bafilomycin A1-treated cells were then transferred into nutrient-rich (DMEM) medium for 20 or 40 min (indicated as 20 and 40). Lysates were prepared and LC3 and tubulin were detected by western analysis. The ratio of LC3-II:tubulin was calculated and is plotted in the graph with a representative western analysis shown. (B) The same fibroblasts as in A were either left in nutrient-rich medium or starved for 2 h. The cells were the processed for immunofluoerescence microscopy as described in the ‘Materials and methods’ section. (C) The per cent overlap between LC3 and LAMP1 from the cells in B was calculated using Imaris for n = 10 cells in all cases. (D) The same fibroblasts as in A were grown in starvation (EBSS) medium for 2 h in the presence of bafilomycin A1. Lysates were prepared and processed for the membrane sealing assay as described in the ‘Materials and methods’ section. Prior to fractionation, some samples were treated with proteinase K (ProK) or with ProK in the presence of 1% Triton X-100 (TX). The fractions, composed of a postnuclear supernatant (PN), low-speed pellet (LP), high-speed pellet (HP) and high-speed supernatant (HS) were probed for LC3. The reduction in LC3-II following proteinase K was ∼50% in lysates from the affected subject. Data are a box-and-whisker plot showing median, interquartile interval, minimum and maximum of n > 3 per measurement from at least three biological collections of an affected subject and at least two independent experiments. Significance was measured by a one-way ANOVA with a Tukey’s post hoc HSD analysis. NS = not significant. **P < 0.01.

Figure 6

The yeast trs23ts-1 variant results in low level of the Trs23 protein and impaired assembly of the TRAPP core. (A) Wild-type and trs23-1 mutant cells were transformed with an empty CEN plasmid (Ø) or a plasmid expressing TRS23 with its own promoter and terminator. Cells were grown at permissive (26°C) or restrictive (37°C) temperature. The trs23ts-1 cells had a growth defect at 26°C and were non-viable at 37°C, which was rescued by introduction of the wild-type plasmid. Shown are two independent colonies for wild-type (top) and trs23-1 (bottom), transformed with empty plasmid (Ø) or plasmid expressing TRS23. (B and C). The interaction of the trs23ts-1 mutant protein with other TRAPP complex subunits is shown in 3D; Trs23 (green), Bet3 (yellow and light pink), Bet5 (magenta), and Trs31 (cyan), associated with Ypt1 (grey). In Trs23, the deleted five amino acids are highlighted in orange. The deleted five amino acids are not in a region of Trs23 that associates with other TRAPP complex proteins (Bet3 and Bet5) and is on the opposite side of its interaction surface with Ypt1. Ribbon 3D diagram was rendered using PyMOL. (C) Focuses on the mutated domain of trs23ts-1 (180° rotation from B) and shows the 214W side chain. (D) Wild-type and trs23-1 mutant cells were transformed with an empty CEN plasmid (Ø) or a plasmid expressing TRS23. Lysates were collected and probed for Trs23 and G6PDH as a loading control. The numbers beneath each lane represent the relative levels of Trs23 compared to control. Quantification of a minimum of three such immunoblots normalized to G6PDH is shown. (E) Wild-type and trs23ts cells expressing Trs31-yEGFP were transformed with a plasmid expressing GST or GST-Bet5. Cell lysates were subjected to GST pulldown followed by immunoblot analysis. The cartoon shows the architecture of core TRAPP complex. Bet5 was revealed using anti-GST, Trs31 was revealed with anti-GFP and the remainder of the subunits were revealed with subunit-specific antibodies. G6PDH was used to ensure equal loading of the lysates (not shown). Molecular size standards are indicated on the right (kDa). Results in D are presented as a box-and-whisker plot showing median, interquartile interval, minimum and maximum of n > 3 per measurement from at least three biological collections.

Figure 7

trs23ts-1 cells display secretory and autophagy defects. (A) trs23ts-1 cells exhibit a general secretion defect at 39°C (right) but not at 25°C (left). S³⁵-labelled proteins from media of cells grown at 25°C or 39°C were precipitated with trichloroacetic acid (TCA) and analysed by SDS-PAGE and Phosphoimager (see ‘Materials and methods’ section). Shown from left to right for each temperature: wild-type, trs23ts-1, trs23ts-1 transformed with a plasmid expressing TRS23, and trs23ts-1 transformed with an empty plasmid (Ø). (B) Wild-type, trs23ts-1 and ypt1-1 were starved in medium lacking nitrogen. Cell survival was determined by vital staining with trypan blue. (C) Cells (wild-type or trs23ts-1) expressing GFP-Atg8 were transformed with an empty CEN plasmid (Ø) or a plasmid expressing TRS23. Transformants were grown in selective medium containing nitrogen (+N₂, left) or incubated for 4 h in medium without nitrogen (−N₂, right). Cell lysates were subjected to immunoblot analysis using anti-GFP (top) and anti-G6PDH (as a loading control). The per cent GFP detected for each condition is shown beneath each lane along with the standard deviation. (D) Cell lysates from C were subjected to an immunoblot analysis using anti-Ape1 and anti-G6PDH (as a loading control) to determine preApe1 (pApe1) and mature Ape1 (mApe1) levels. The per cent mApe1 detected in each condition is indicated beneath each lane along with the standard deviation. (E) Wild-type and trs23ts were transformed with a 2-μ plasmid for expression of GFP-Snc1-PEM and grown at 26°C. GFP-Snc1-PEM levels were determined by immunoblot analysis with anti-GFP antibody. G6PDH was included as a loading control. The level of GFP-Snc1-PEM relative to wild-type is indicated beneath each lane along with the standard deviation. (F) Wild-type (top) and trs23ts (bottom) cells expressing endogenously-tagged Sec61-mCherry and GFP-Snc1-PEM from a plasmid were grown as in E and cells were visualized by confocal fluorescence microscopy. Shown from left to right: DIC (cell contour), GFP channel, mCherry channel, merge. The percentage of cells with intracellular GFP-Snc1-PEM (±SD) and per cent co-localization of GFP-Snc1-PEM with Sec61-mCherry are indicated to the right of the fluorescence micrographs. Scale bar = 1 µm. Molecular size standards are indicated to the right of the gels and immunoblots in A, C, D and E. Results in this figure represent three independent experiments.