Bi-allelic variants in INTS11 are associated with a complex neurological disorder

Abstract

The Integrator complex is a multi-subunit protein complex that regulates the processing of nascent RNAs transcribed by RNA polymerase II (RNAPII), including small nuclear RNAs, enhancer RNAs, telomeric RNAs, viral RNAs, and protein-coding mRNAs. Integrator subunit 11 (INTS11) is the catalytic subunit that cleaves nascent RNAs, but, to date, mutations in this subunit have not been linked to human disease. Here, we describe 15 individuals from 10 unrelated families with bi-allelic variants in INTS11 who present with global developmental and language delay, intellectual disability, impaired motor development, and brain atrophy. Consistent with human observations, we find that the fly ortholog of INTS11, dIntS11, is essential and expressed in the central nervous systems in a subset of neurons and most glia in larval and adult stages. Using Drosophila as a model, we investigated the effect of seven variants. We found that two (p.Arg17Leu and p.His414Tyr) fail to rescue the lethality of null mutants, indicating that they are strong loss-of-function variants. Furthermore, we found that five variants (p.Gly55Ser, p.Leu138Phe, p.Lys396Glu, p.Val517Met, and p.Ile553Glu) rescue lethality but cause a shortened lifespan and bang sensitivity and affect locomotor activity, indicating that they are partial loss-of-function variants. Altogether, our results provide compelling evidence that integrity of the Integrator RNA endonuclease is critical for brain development.

Keywords: CPSF3L; Drosophila; INTS11; brain atrophy; dIntS11; delayed language development; developmental delay; impaired motor development; intellectual disability.

Figure 1

Most variants in INTS11 are predicted to be deleterious (A) Domain structure of human INTS11 protein. INTS11 is 606 amino acid (aa) long and contains three domains: metallo-β-lactamase (light blue), β-CASP (orange), and C-terminal domain (CTD) (tan). Variants identified in this study are indicated above/below the protein as dots and are not clustered in a specific domain. (B) Table summarizing the nature of the variants and their predicted consequence on protein function. Coding changes are described using the GenBank: NM_001256456.1 transcript. Variants modeled in this study are highlighted in yellow. Variants predicted to be damaging based on SIFT or PolyPhen are highlighted in red (SIFT, Sorting Intolerant From Tolerant; PolyPhen, Polymorphism Phenotyping; CADD, Combined Annotation Dependent Depletion). (C) INTS11 is predicted to cause a recessive disease. INTS11 is not constrained based on the presence of LoF variants in gnomAD, resulting in a low probability-of-LoF-intolerance (pLI) score and missense constraint Z score. Based on gnomAD data and the DOMINO algorithm, variants in INTS11 are likely to cause a recessively inherited disease.

Figure 2

Loss-of-function mutations in IntS11 in flies cause lethality (A) Genomic location of the genomic duplication (Dp) was used to rescue IntS11 mutants, and deficiency construct (Df) was used to uncover the gene for complementation analysis. (B) Strategy to generate the Kozak-Gal4 insertion in dIntS11 locus. Using CRISPR-Cas9-mediated genome editing, we inserted the Kozak-Gal4 cassette into the IntS11 locus while removing the entire coding region of the gene. (C) Strategy to study the effects of variant IntS11 in flies. Combining IntS11^Kozak-Gal4 with UAS-mCherry.NLS allows the determination of the expression pattern. The Kozak-Gal4 insertion also generates a null allele to investigate mutant phenotypes and to perform rescue assays using UAS-dIntS11 or UAS-hINTS11. We also ectopically express dIntS11 or hINTS11 reference (Ref) using a ubiquitous GAL4 driver to assess the toxicity of overexpression in vivo. (D) The homozygous IntS11^Kozak-Gal4 flies are lethal over a genomic deficiency covering the dIntS11 locus. Reintroduction of dIntS11 with a Dp construct or Kozak-Gal4-driven overexpression of dIntS11 rescues the lethality phenotype of IntS11^Kozak-Gal4/Df flies, while overexpression of human reference cDNA does not rescue the lethality. (E) Overexpression of UAS-dIntS11 or UAS-hINTS11 does not cause any change in viability. Three different temperatures are used to control the level of overexpression. The expression level of the UAS construct is lowest at 18°C, highest at 29°C, and intermediate at 25°C.

Figure 3

dIntS11 is expressed in neurons and glia in the CNS (A) The expression pattern of dIntS11 in the L3 larval CNS (left) and adult brain (right) is visualized by dIntS11^Kozak-Gal4-driven expression of mCherry.NLS. Scale bars: 100 μm. (B) L3 larval CNS (top two panels) and adult brain (bottom two panels) samples expressing dIntS11^Kozak-Gal4-driven mCherry.NLS are co-stained with markers for neurons (Elav) or glia (Repo). Colocalization images generated using IMARIS indicate that mCherry is colocalized with both neurons (Elav) or glia (Repo). Scale bars: 100 μm.

Figure 4

Glial IntS11 knockdown reduces viability, and escapers have a short lifespan with climbing defects (A) Real-time RT-PCR data show that two different RNAis (GD and KK) driven by a weak ubiquitous driver (Da-Gal4) reduce dIntS11 mRNA levels to about 20% of the endogenous levels in control animals at 25°C. Data are means +SEM. Unpaired test (^∗∗∗∗p < 0.0001). (B) Glial knockdown of dIntS11 with both GD- and KK-RNAi reduces the viability of adult flies, as shown by lower-than-expected genotypic survival ratios into adulthood. Repo-Gal4>UAS-GD-RNAi or UAS-KK-RNAi flies were compared to Repo-Gal4>UAS-Luciferase-RNAi (control) flies. Data are means + SEM. Unpaired test (^∗p < 0.05, ^∗∗∗p < 0.001). (C) Summary of lethality phenotype of IntS11 knockdown using various Gal4 drivers. Both GD- and KK-RNAi lines reduce viability when driven by a weak ubiquitous driver (Da-Gal4), and no viable flies were observed when driven by a strong ubiquitous driver (Act-Gal4). Pan-neuronal knockdown of IntS11 with elav-Gal4-driven RNAis did not cause any apparent phenotypes, while repo-Gal4-driven glial knockdown was semi-lethal for both RNAi lines. (D) The lifespan of glial IntS11 knockdown flies. Data are the probability of survival at different ages. Log rank (Mantel-Cox) test (^∗∗∗∗p < 0.0001). (E) Climbing speed for glial IntS11 knock down flies at 10- and 40-day post eclosion. Data are means +SEM. Unpaired test (^∗∗p < 0.01, ^∗∗∗∗p < 0.0001).

Figure 5

Two of the variants are strong loss-of-function alleles and five of the variants are weak loss-of-function alleles (A) Summary of missense variants, their fly counterparts, and their effect on the lethality phenotype of dIntS11^Kozak-Gal4. pArg17Leu and pHis414Tyr variants do not rescue lethality, arguing that they are strong loss-of-function alleles. (B) The lifespan of flies rescued with wild-type or variant cDNA. Data are the probability of survival at different ages. Log rank (Mantel-Cox) test (^∗∗∗∗p < 0.0001).

Figure 6

Reduced level of variant cDNA expression reveals locomotor defects in rescue flies (A) Schematic representation of Gal80^ts-mediated Gal4-UAS system inhibition. Gal80^ts binds to Gal4 at a restrictive temperature (18°C) and blocks the transcriptional activation domain of Gal4, leading to the inhibition of downstream UAS constructs. At permissive temperature (29°C), Gal80^ts degrades, and Gal4 can drive the expression of downstream UAS constructs. Intermediate temperatures allow fine-tuning of UAS construct expression. (B) Summary of lethality phenotype rescued with wild-type dIntS11 at different temperatures and the observed/expected ratios at 25°C. Data are means + SEM. Unpaired test (^∗p < 0.05). (C) Bang sensitivity of null mutant flies rescued with IntS11 variants at 25°C measured using the time to recover after 15 s of vortex. Kruskal-Wallis test was followed by a Dunn’s test. Data are means + SEM (^∗p < 0.05, ^∗∗p < 0.01). (D) Locomotor activity of null mutant flies rescued with IntS11 variants at 25°C measured by DAM assay. Kruskal-Wallis test was followed by a Dunn’s test. Data are means +SEM (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001).