Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease

Abstract

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9^-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Keywords: Charcot-Marie-Tooth disease; DExD/H-box RNA helicases; DHX9; DNA damage repair; R-loops; allelic series; epilepsy; homologous recombination; neurodevelopmental disorders; neuropathy.

Graphical abstract

Figure 1

Missense and LoF tolerance in the DDX/DHX superfamily and DHX9 expression (A) Relationship between the pLoF and missense constraints among known or candidate DDX/DHX genes associated with disease phenotypes. Genes linked to dominant disease traits are shown in red, those linked to recessive traits are shown in blue, and those linked to mixed dominant and recessive traits are shown in orange. The solid line indicates linear regression. The dashed line shows the top LOEUF decile. The y axis shows LOEUF, and the x axis shows missense Z scores (gnomAD v2.1.1). (B) Relationship between pLoF and missense constraint among all DDX/DHX genes. DHX9 is indicated by a red dot. Paralogs DHX30 and DDX3X are indicated by blue dots. The solid line indicates linear regression. The dashed line shows the top LOEUF decile. The y axis shows LOEUF, and the x axis shows missense Z scores (gnomAD v2.1.1). (C) DHX9 mRNA expression in human adult tissues from the GTEx project. Nervous system tissues are highlighted in gray. The y axis shows transcripts per million (TPM). (D) Average mRNA expression of known or candidate DDX/DHX genes associated with disease phenotypes in the developing nervous system from BrainSpan. DHX9 is indicated by red dots. Paralogs DHX30 and DDX3X are indicated by blue dots. The y axis shows log2 reads per kilobase million (RPKM), and the x axis shows the developmental stage. Abbreviations: pcw, post-conception weeks; mo, months; yrs, years.

Figure 2

Pedigrees, photographs, and brain imaging of individuals with candidate disease-causing DHX9 variants Pedigrees, DHX9 genotypes, and representative brain MRIs of individuals with NDDs are shown above the black line. Note that family 3 and family 12 share the recurrent allele c.3497G>C (p.Arg1166Pro). Individuals, genotypes, and representative clinical images and leg-muscle MRIs of individuals with CMT are shown below the black line. The yellow arrow indicates a thin corpus callosum. Red arrows indicate cerebellar atrophy. Yellow arrowheads show enlargement of the ventricles, and yellow asterisks (brain MRI, second image from the left) show reduced white-matter volume. The photograph shows the pes cavus and hammer-toe deformity in family 15, affected by CMT. Yellow asterisks (leg MRI) highlight fatty infiltration of the lower-leg musculature, consistent with CMT. Black pedigree symbols indicate NDDs, whereas gray pedigree symbols indicate CMT. Individuals for whom limited clinical details were available were excluded from the figure (see supplemental notes).

Figure 3

Location of DHX9 variants (A) Diagram of DHX9 mRNA shows the location of NDD-associated (white) and CMT-associated (black) variants. (B) Diagram of DHX9 (UniProt: Q08211) shows functional domains, including dsRBD1 and dsRBD2, the MTAD, helicase domains, HA2, the OB fold, and the RGG box. Protein domains were obtained from UniProt. The sequence of the nuclear localization signal is magnified, and the two key residues (Lys1163 and Arg1166) are underlined. DHX9’s protein-tolerance landscape, calculated by Metadome, is shown at the bottom.

Figure 4

HPO analysis of the DHX9 cohort (A) Gap statistic curve for the DHX9 cohort. The gap statistic is displayed on the y axis, and the number of clusters tested is on the x axis. The point on the curve where the slope changes from a trend of higher to lower (i.e., additional clusters do not add as much to the gap statistic) was chosen as the optimal number of clusters (k = 4). (B) HAC and visualization of quantitative phenotypic similarity allow refinement of genotype-phenotype correlations in the DHX9 cohort. The dendrogram shown at the top and to the left of the heatmap is based on HAC analysis of the dissimilarity matrix produced from Resnik semantic similarity scores and with k = 4. Unique clusters are represented by different colors, and variants found in individual probands are labeled on top of and to the right of the heatmap. Within the heatmap, dark red indicates a higher similarity, whereas dark blue indicates a lower similarity. A key is provided on the right. (C) Magnified dendrogram showing unique clusters and group characteristics.

Figure 5

Subcellular localization of WT DHX9 and representative variant proteins (A) Subcellular localization of EGFP-tagged WT DHX9, NLS p.Lys1163Arg (severe NDD), truncating p.Arg229Ter (mild NDD), and CMT p.Ala1255Thr proteins in MCF-7 and PC-3 human cells. Nucleolar loci were co-stained by the FBL marker, and DNA was stained by DAPI. Scale bar: 10 μm. (B) Endogenous localization of DHX9 in fibroblasts from the proband with severe NDD and the p.Lys1163Arg NLS variant as well as the unaffected father. Scale bar: 40 μm. (C) Staining of R-loop formation by the S9.6 marker and of DSBs by the γ-H2AX marker in MCF-7 cells producing the WT or p.Arg141Gln protein. Scale bar: 10 μm. Also see Figures S7 and S8.

Figure 6

Disruption of Dhx9 in mice causes behavioral and neurological abnormalities (A–C) Dhx9^−/− mice were hypoactive in response to a novel environment (A: decreased distance traveled; B: decreased speed) in an open field and to decreased rearing (C) upon the first introduction to a novel home cage. However, male Dhx9^−/− mice showed more rearing activity with lights off during the active phase in the home cage. This increased vertical activity was evident in both male and female Dhx9^−/− mice in the latter half of the active phase. Shaded areas encompass the period between lights off and lights on (18:00–06:00) and represent the mean ± SEM. (D and E) Both 2-paw and 4-paw grip strength were reduced in Dhx9^−/− mice (D). Lower grip strength was predicted by the lower body weight in linear regression analysis (E). (F) 20% of Dhx9^−/− mice showed tremors in the SHIRPA analysis. (G and H) Both an absent auditory response curve in the auditory brainstem response (G) and decreased acoustic startle reactivity (H) indicated deafness in Dhx9^−/− mice. (I–K) The body weight of Dhx9^−/− mice was lower than that of WT mice (I). Dhx9^−/− mice had lower cumulative food intake and lost more weight over a 21 h period than WT mice after transferring to a new home cage. #p < 0.05, male +/+ vs. −/−; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001, +/+ vs. −/−. Data represent the mean ± SD.