Mascibroda LG et al. (2022), INTS13 variants causing a recessive development...

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Nat Commun 2022 Oct 13;131:6054. doi: 10.1038/s41467-022-33547-8.

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INTS13 variants causing a recessive developmental ciliopathy disrupt assembly of the Integrator complex.

Mascibroda LG , Shboul M , Elrod ND , Colleaux L , Hamamy H , Huang KL , Peart N , Singh MK , Lee H , Merriman B , Jodoin JN , Sitaram P , Lee LA , Fathalla R , Al-Rawashdeh B , Ababneh O , El-Khateeb M , Escande-Beillard N , Nelson SF , Wu Y , Tong L , Kenney LJ , Roy S , Russell WK , Amiel J , Reversade B , Wagner EJ .

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Oral-facial-digital (OFD) syndromes are a heterogeneous group of congenital disorders characterized by malformations of the face and oral cavity, and digit anomalies. Mutations within 12 cilia-related genes have been identified that cause several types of OFD, suggesting that OFDs constitute a subgroup of developmental ciliopathies. Through homozygosity mapping and exome sequencing of two families with variable OFD type 2, we identified distinct germline variants in INTS13, a subunit of the Integrator complex. This multiprotein complex associates with RNA Polymerase II and cleaves nascent RNA to modulate gene expression. We determined that INTS13 utilizes its C-terminus to bind the Integrator cleavage module, which is disrupted by the identified germline variants p.S652L and p.K668Nfs*9. Depletion of INTS13 disrupts ciliogenesis in human cultured cells and causes dysregulation of a broad collection of ciliary genes. Accordingly, its knockdown in Xenopus embryos leads to motile cilia anomalies. Altogether, we show that mutations in INTS13 cause an autosomal recessive ciliopathy, which reveals key interactions between components of the Integrator complex.

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Species referenced: Xenopus laevis
Genes referenced: arl13b bbof1 ccdc9 cfap53 ddx59 ints11 ints13 ints4 ints9 lgals4.2 mcc ofd1 rpe sacs tmem231 tuba4b
???displayArticle.morpholinos??? ints13 MO2 ints13 MO3

???displayArticle.disOnts??? ciliopathy
???displayArticle.omims??? MOHR SYNDROME
Phenotypes: Xla Wt + ints13 MO (Fig. 7b, r1c2) [+]

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	Fig. 1: Identification of two families segregating recessive INTS13 variants. a Pedigree of Family 1 (top): affected individuals (II.4 and II.5) were born to first-cousin parents (I.1 and I.2) with three unaffected children (II.1-II.3). Double lines indicate a consanguineous marriage. Pedigree of Family 2 (bottom): affected individuals (II.2 and II.4) were born to parents with two unaffected children (II.1 and II.3). b Homozygosity mapping of Family 1 delineated five candidate loci totaling 46 cM on chromosomes 6, 8, 12, 13, and 16. c Schematic representation of human INTS13 located on chromosome 12 (ch12p13.2-p11.22) consisting of 17 exons. Locus capture followed by massive parallel sequencing identified a homozygous frameshift mutation as a disease-causing mutation in Family 1. The c.2004delA (p.K668Nfs*9) mutation is caused by a single base pair deletion in exon 16 (marked in brown), resulting in a frameshift and premature termination codon (PTC) which alters nine amino acids (marked in red) and deletes the last 31. In Family 2, the c.1955C > T (p.S652L) mutation is caused by a single base pair substitution in exon 16 (marked in green). d Amino acid alignment of the C-terminus of the INTS13 protein shows that both mutations occur at highly conserved residues. Red and yellow shading indicates regions of conservation. Source data are provided as a Source Data file.
	Fig. 2: Characterization of INTS13 variants using patients’ cells. a RT-qPCR shows that endogenous INTS13 transcript levels were significantly reduced relative to control cells in primary dermal fibroblasts of affected individual II.4 from Family 1. Data are mean + /- SD, n = 3 replicate cell samples. Statistical significance was calculated using a two-tailed unpaired t-test. p = 0.000223. b Primary dermal fibroblasts were treated with cycloheximide (CHX) (100 g/ml). Following treatment, qRT-PCR was done at 8 h and shows that INTS13 mRNA level was increased in treated cells indicating a non-sense mediated decay. Data are mean ± s.e.m., P < 0.05; P < 0.005; *P < 0.001; one-tailed Student’s t-test. c Western blot analysis of primary dermal fibroblasts of affected individual II.4 from Family 1 using two polyclonal antibodies with antibody directed against the C-terminus of INTS13 (C) and the second antibody directed against the center of INTS13 (M). Actin was used as a loading control and molecular weight markers shown are in kilodaltons. d RT-qPCR shows that INTS13 transcript levels were significantly reduced in primary dermal fibroblast cells of affected individual II.4 from Family 2 relative to control cells. Data are mean + /- SD, n = 3 replicate cell samples. Statistical significance was calculated using a two-tailed unpaired t-test. p = 0.003833. e Western blot analysis of primary dermal fibroblasts of affected individual II.4 from Family 2 shows reduced levels of INTS13 protein compared to control cells. f Western blot analysis of primary dermal fibroblasts from individual II.4 from Family 2 treated with MG132. Treated cells show a level of INTS13 protein comparable to control cells. indicates a non-specific signal. Multiple western blots were developed (> 3), and the results shown are representative of the data obtained. g Multiciliated airway cells were obtained from nasal biopsies of three members of Family 1: the unaffected carrier mother (Con.) and the two affected children (II.4 and II.5). Probing for acetylated α-tubulin shows significantly shorter, less dense, and disorganized cilia in the affected individuals compared to the mother’s cells. scale bar is 5 μm. h The average length of nasal cilia is significanty reduced in the two affected sisters compared to that of their mother. Data are mean ± s.e.m., ***P < 4 × 10−9, n ≥ 31 cells for each patient in one biological replicate. Source data are provided as a Source Data file.
	Fig. 3: INTS13 associates with the INTS4/9/11 heterotrimer. a Yeast two hybrid screening for interactors of INTS13. Haploid strains of yeast expressing each individual Integrator subunit fused to the Gal4 activating domain (AD) were mated with strains expressing INTS13 fused to the DNA binding domain (BD). The first and third panels show mated yeast grown on selective media as a positive control (media lacking leucine, tryptophan, and uracil). The remaining panels are of yeast plated on restrictive media additionally lacking histidine to test for protein-protein interactions and with 3AT (3-amino-1,2,4-triazole) to test the strength of the interaction. No interaction was observed between INTS13 and any other individual subunit (second panel). When INTS9 and INTS11 were expressed in trans with INTS13, there was a positive interaction with INTS4 (fourth panel). b Point mutants of INTS4, INTS9, and INTS11 known to disrupt their interactions were used in yeast two hybrid to demonstrate that the formation of the INTS4/9/11 heterotrimer is necessary for INTS13 association. General locations of the mutations are indicated with arrows on the schematic. INTS13 is fused to the activating domain in the rest of the yeast two hybrid assays shown in this study. c SDS PAGE gel of fractions from gel filtration column of co-expressed full-length INTS4, INTS9, INTS11 and INTS13. The sample was first purified by nickel affinity chromatography. INTS4 carried an N-terminal His-tag, while the other three proteins were untagged. The positions of the fractions are indicated at the top of the gel and molecular weight markers shown are in kilodaltons. Multiple purifications were conducted (> 3), and the gel shown is representative of the results attained. d Consecutive 200-amino acid truncations of INTS13 were tested for interaction with INTS4/9/11. In modified yeast two-hybrid assays, the only positive interactions seen are full length INTS13 and the last ~200 residues, 501-706. The patient mutations are marked on the schematic and occur in this region. Loss of interaction with INTS4/9/11 was observed using the mutant versions of INTS13. Source data are provided as a Source Data file.
	Fig. 4: INTS13 patient variants disrupt interactions between the INTS10/13/14 module and the Integrator complex. a Immunoprecipitation of FLAG-INTS13 from nuclear extract derived from HEK-293T cells stably expressing FLAG epitope tagged human INTS13-wild type (wt), Family 1 INTS13 mutant (F1), or Family 2 INTS13 mutant (F2). Nuclear extract derived from naïve HEK-293T cells not expressing any FLAG epitope was used as a control. Left panel: IP-LC/MS was done for each sample, and quantitation is shown as a heat map. Results from the MS experiments were quantified from triplicate experiments, while the IP/WB was conducted in duplicate with representative images shown in the panel. The color scale shown denotes the normalized spectral counts for each protein (see methods). Right panel: western blot analysis of either nuclear extract input or anti-FLAG immunoprecipitation using human Integrator antibodies with molecular weight markers shown are in kilodaltons. b Results shown are from parallel experiments conducted in a manner identical with panel (a) with the exception that nuclear extract was derived from Drosophila S2 cells expressing FLAG-dINTS13-wt or FLAG-tagged dINTS13 proteins where patient mutations were introduced into homologous regions of the Drosophila INTS13 cDNA. Results from the MS experiments were quantified from triplicate experiments, while the IP/WB was conducted in duplicate with representative images shown in the panel. c Modified yeast two hybrid analysis where human INTS14 is expressed as a Gal4-DNA binding domain fusion, human INTS13 wt or mutant is expressed as a Gal4-Activation domain fusion, and human INTS10 is expressed in trans. Dilutions of yeast are plated on both permissive (left) and restrictive (right) media. Empty activation domain vector is used as a negative control. d Schematic of the Integrator CM associating with the remainder of Integrator subunits that is bridged to the INTS10/13/14 module through the C-terminus of INTS13, which is disrupted by OFD2 mutations. Source data are provided as a Source Data file.
	Fig. 5: INTS13 depletion leads to a loss of primary cilia and broad transcriptional perturbation of ciliary genes. a Western blot of whole cell lysates from RPE cells treated with non-targeting control siRNA (Con.) or either of two siRNA targeting INTS13 (INTS13-1, INTS13-2). Lysates were probed for either INTS13 or tubulin as a loading control and molecular weight markers shown are in kilodaltons. RNAi-depletion and Western blotting was repeated in two independent experiments with a representative image shown. b Immunofluorescence imaging of RPE cells treated with siRNAs as in (a) to visualize primary cilia (PC). DAPI staining in blue indicates the nuclei, γ-tubulin staining is shown in red to visualize the basal body, and ARL13B staining is shown in green to visualize the ciliary axoneme. Representative images show disrupted primary cilia that occurred when INTS13 was targeted by RNAi. The scale bar for the merged image is set at 3 μm while the scale bar in the figure inset present in the center is 1 μm. c Images from (b) were quantified for each condition and graphed as a percentage of the number of control-treated cells with PC (data are mean +/- SD, con. n = 3785 cells, 13-1 n = 2969 cells, 13-2 n = 2675 cells. Raw calculations for each condition were divided by the control mean to adjust control to 1). Statistical significance was calculated using a one-way ANOVA with Tukey’s multiple comparisons test. Control vs INTS13-1 and INTS13-2 p < 0.0001, and INTS13-1 vs INTS13-2 p = 0.0024. d RNA sequencing was done on siRNA treated RPE cells in triplicate. Genes with significant changes in expression are shown by color indicating the log2 fold change. e Results from (d) were used for gene ontology analysis: the top five significant results are shown for upregulated and downregulated genes. All five GO terms for upregulated genes are related to cilia. f Quantitative RT-PCR validating three ciliary genes with expression changes identified using RNA-seq. Results are quantified from independent biological replicates (n = 3, data are mean + /- SD). Statistical significance was calculated using multiple unpaired t-tests with a Holm-Sidak correction. For 13-1 compared to control, CFAP206 p = 0.00287, CFAP53 p = 0.02511, and BBOF1 p = 0.0221. For 13-2 compared to control, CFAP206 p = 0.01878, CFAP53 p = 0.00049, and BBOF1 p = 0.00013 p < 0.05 p < 0.01, p < 0.001, **p < 0.0001 respectively. Source data are provided as a Source Data file.
	Fig. 6: Human wild-type INTS13, but not mutant INTS13-F1, rescues spermatogenesis defects of Drosophila mutant ints13 males. a–j Male germline expression of wild-type, full-length human INTS13 (INTS13-wt), but not a truncated form (INTS13-F1), restored perinuclear dynein in Drosophila asun primary spermatids (a–d; quantified in e) and spermatocytes (f–i; quantified in j). Representative G2 spermatocytes and immature spermatids stained for dynein heavy chain (green) and DNA (blue) are shown. Bar graphs depict percentages of spermatids and spermatocytes showing properly localized dynein heavy chain. k–o Male germline expression of wild-type, full-length human INTS13, but not INTS13-F1, restored nucleus-centrosome coupling in asun primary spermatocytes (k–n; quantified in o). Representative prophase I spermatocytes stained for β-tubulin (green) and DNA (blue) are shown. Bar graphs depict percentages of spermatocytes showing normal nucleus-centrosome coupling. Data are mean ± s.e.m. *P < 0.0001, one-tailed Student’s t-test. e, j n > 10 flies were used with n > 584 spermatids or spermatocytes scored. o n > 10 flies were used with n > 120 spermatocytes scored. INTS13-F1 is encoded by INTS13 c.2004delA. Scale bars, 10 µm. Source data are provided as a Source Data file.
	Fig. 7: INTS13 is required for embryonic development and ciliogenesis in Xenopus laevis. a Western blot showing that the INTS13 MO was able to deplete INTS13 protein in Xenopus embryos and molecular weight markers shown are in kilodaltons. b In comparison to control embryos, morpholino (MO)-mediated knock down of INTS13 in Xenopus leads to severe developmental defects including a small head, big belly, short and down curved body axes, cardiac edema and massive developmental delay. Scale bar represents 500 μM. c In situ hybridization using a specific probe for the multi-ciliated cell (MCC) marker ccdc9 shows a reduction in ccdc9 expression in MCC of morphants compared to control embryos. d Whole mount antibody staining for acetylated α-tubulin shows a punctate pattern similar to ccdc9. The puncta have a more condensed and stronger signal in control embryos. A higher magnification view of the MCC shows marked reduction in cilia compared to controls. b–d n > 60 control or morphants embryos were used for three sets of independent injections. Scale bar represents 50 μM. e Low magnification SEM imaging of Xenopus MCC at embryonic stage 28. MCCs of a control embryo possess multiple, long cilia, whereas the morphant MCCs display fewer cilia per cell which appear to be disorganized. f High magnification SEM imaging of Xenopus MCC at embryonic stage 28. Ciliary length seems unperturbed in morphant embryos, while the average number of cilia on individual MCCs is clearly reduced by INTS13 knockdown. The average MCC in morphants have less than half of the number of cilia per cell compared to control, as shown in the bar graph. g The ultrastructure of the cilia in morphants appears to show a normal 9 + 2 structure as imaged by TEM. e, f, n = 3 control or morphant tadpoles were processed for SEM and n > 30 multiciliated cells were examined for the number of cilia present. g n = 5 cilia were examined by TEM. p < 0.05, p < 0.01, p < 0.001, **p < 0.0001, respectively. Source data are provided as a Source Data file.
	ccdc9 (coiled-coil domain containing 9 ) gene expression in a X. laevis embryo, NF stage 28, assayed via in situ hybridization, lateral view, anterior left, dorsal up.
	Tuba4a (acetylated tubulin) expression in a X. laevis embryo, NF stage 28, assayed via immunohistochemistry, lateral view, anterior left, dorsal up.
	Supplementary Fig 6. Expression of ints13 during Xenopus development. a Detailed expression pattern of ints13 in Xenopus embryos analyzed by WISH and whole mount immunostaining. ints13 has both maternal and zygotic contribution. At 4 cell stage, ints13 transcripts are localized within the animal half. At stage 10, its expression covers the whole embryo except the blastopore. At stage 17, ints13 is observed in neural tube and neural folds. b Lateral view, anterior is right stage 22 ints13 starts to be enriched in brain, branchial arches (ba), pronephros (pn), eye vesicles (ev), ear (e), skin cells (sk) and somites (so). Lateral view, anterior is right of the embryo at stage 28, ints13 expression is also detected in ear (e), skin cells (sk) and pronephros (pn). Correlation of transcripts localization of ints13 protein at stage 28 by wholemount embryo antibody staining using a custom polyclonal INTS13 (α-M) antibody, Lateral view, anterior is left.

References [+] :

Adly, Ciliary genes TBC1D32/C6orf170 and SCLT1 are mutated in patients with OFD type IX. 2014, Pubmed