De Novo Loss-of-Function Mutations in USP9X Cause a Female-Specific Recognizable Syndrome with Developmental Delay and Congenital Malformations

Abstract

Mutations in more than a hundred genes have been reported to cause X-linked recessive intellectual disability (ID) mainly in males. In contrast, the number of identified X-linked genes in which de novo mutations specifically cause ID in females is limited. Here, we report 17 females with de novo loss-of-function mutations in USP9X, encoding a highly conserved deubiquitinating enzyme. The females in our study have a specific phenotype that includes ID/developmental delay (DD), characteristic facial features, short stature, and distinct congenital malformations comprising choanal atresia, anal abnormalities, post-axial polydactyly, heart defects, hypomastia, cleft palate/bifid uvula, progressive scoliosis, and structural brain abnormalities. Four females from our cohort were identified by targeted genetic testing because their phenotype was suggestive for USP9X mutations. In several females, pigment changes along Blaschko lines and body asymmetry were observed, which is probably related to differential (escape from) X-inactivation between tissues. Expression studies on both mRNA and protein level in affected-female-derived fibroblasts showed significant reduction of USP9X level, confirming the loss-of-function effect of the identified mutations. Given that some features of affected females are also reported in known ciliopathy syndromes, we examined the role of USP9X in the primary cilium and found that endogenous USP9X localizes along the length of the ciliary axoneme, indicating that its loss of function could indeed disrupt cilium-regulated processes. Absence of dysregulated ciliary parameters in affected female-derived fibroblasts, however, points toward spatiotemporal specificity of ciliary USP9X (dys-)function.

Figure 1

Identified De Novo USP9X Loss-of-Function Mutations (A) Detailed view of the USP9X (GenBank: NM_001039590.2) region and the reported deletions. (B) Overview of USP9X including UBL and catalytic domain and the location of reported mutations according to their relative position at the protein level. The reported amino acid substitution is located within the catalytic domain. (C) RNA was extracted from both control and affected female (c.3028−2A>G [p.(?)]) fibroblasts cultured under normal conditions or in the presence of cycloheximide (CHX) to inhibit NMD. After cDNA synthesis and PCR, agarose gel analysis showed two different product sizes generated from the c.3028−2A>G transcript but only one from the control fibroblast transcript. Excision and sequencing of the additional band revealed that the aberrant USP9X transcript lacked exon 21. The level of the aberrant transcript was increased 4-fold when fibroblasts were treated with cycloheximide, confirming that the aberrant transcript was indeed subjected to NMD. (D) USP9X expression is depleted in female cell lines harboring loss-of-function alleles. Relative qPCR analysis of USP9X mRNA and relative quantification of immunoblot analysis of USP9X protein derived from female and male control cell lines and from affected female cell lines. n = the number of individual cell lines analyzed. Each cell line analyzed in quadruplicate. Error bars represent SDs. Asterisk (^∗) indicates significantly different to female controls, p < 0.05 by Student’s t test.

Figure 2

Clinical Characteristics of Females with De Novo USP9X Loss-of-Function Mutations (A) Frontal and lateral photographs of females with de novo mutations in USP9X. Shared facial characteristics include facial asymmetry, prominent forehead, bitemporal narrowing, short palpebral fissures, low nasal bridge, prominent nose with flared alae nasi from adolescence age, thin upper lip, smooth and long philtrum, hanging full cheeks in early childhood, and low-set, posteriorly rotated, and dysplastic ears with attached lobule. (B) Photographs of the hands of seven affected females. Shared characteristics include ulnar deviation of 5^th digit, tapered fingers, short 4^th and 5^th metacarpals, and post-axial polydactyly (simian crease present but not shown). (C) Photographs of the feet of five affected females. Shared characteristics include hallux valgus and sandal gap (pes cavus present but not shown). (D) Observed Blaschko lines of female 3, indicative for 11 of the affected females, suggestive of different X-inactivation pattern between tissues (functional mosaicism).

Figure 3

Representative MRI Images from Females 1, 2, 3, 7, and 16 with De Novo USP9X Loss-of-Function Mutations (A–D) Female 1 (2 years): MRI T2 axial (A, B) and sagittal (C) and T1 axial (D) sections show brachycephaly, mild enlargement of the lateral and 3^rd ventricles; mild hypoplasia of cerebellar vermis and left cerebellar hemisphere; enlarged IV ventricle and cisterna magna with small retrocerebellar cyst; thin brain stem and mesencephalon; relatively small frontal lobes with somewhat simplified gyration; and short hypoplastic corpus callosum (both rostrum and splenium). (E–H) Female 2 (1.5 years): MRI T2 axial (E, F) and T1 sagittal (G) and coronal (H) sections show enlargement of the lateral ventricles, mild hypoplasia of cerebellar vermis and left cerebellar hemisphere; enlarged cistern magna; thin corpus callosum, pons, mesencephalon, and brain stem; and broader and underdeveloped frontal gyri. (I–L) Female 3 (11 years): MRI T2 axial (I, J) and T1 sagittal (K) and axial (L) sections show asymmetric enlargement of the lateral ventricles; simplified convolutions of the frontal lobe gyri; hypoplasia of cerebellar vermis and left hemisphere; large cisterna magna and retrocerebellar cyst; and thin corpus callosum with hypoplasia of the rostrum. (M–P) Female 7: MRI T2 axial (M), T1 axial (N), T1 sagittal (O), and coronal FLAIR (P) sections show macrocephaly; enlargement of the lateral and 3^rd ventricles with an interhemispheric cyst; dysplastic cerebellar hemispheres; dysplasia of the cerebellar vermis which is uplifted, with a high position of the tentorium and a large posterior fossa, typical of Dandy-Walker malformation; and thin and hypoplastic corpus callosum. (Q–T) Female 16 (2 years): MRI T2 axial (Q, R, S) and T1 coronal (T) sections show enlarged lateral ventricles; irregular gyri of the cerebral cortex with irregular depth of the sulci in frontal and perisylvian areas; small heterotopic nodule of gray matter (arrow) and thin and hypoplastic corpus callosum (both rostrum and splenium); hypoplasia of the anterior cerebellar vermis and left cerebellar hemisphere; enlarged cisterna magna and arachnoidal cyst surrounding the cerebellum, especially at the left side; and mild hypoplasia of pons and brain stem. This female was identified with Sanger sequencing based on these brain abnormalities in combination with ID, dysmorphic features, and congenital abnormalities.

Figure 4

USP9X Localizes to the Primary Cilium To induce ciliogenesis, control and affected female fibroblasts, matched for gender and age, were starved for 48 hr prior to immunofluorescence labeling. Endogenous USP9X is detected along the length of the axoneme of primary cilia, using an antibody against its N terminus (N-Term, shown in green) as compared to the ciliary markers RPGRIP1L (pink, denoting the ciliary transition zone at the base of the cilium) and acetylated alpha-tubulin (red, marking the ciliary axoneme). DAPI (blue) stains the nuclei; scale bars represent 10 μm. Ciliated fibroblasts derived from affected females are shown here, USP9X localization in control fibroblasts is shown in Figure S2.