WDR11, a WD protein that interacts with transcription factor EMX1, is mutated in idiopathic hypogonadotropic hypogonadism and Kallmann syndrome

Abstract

By defining the chromosomal breakpoint of a balanced t(10;12) translocation from a subject with Kallmann syndrome and scanning genes in its vicinity in unrelated hypogonadal subjects, we have identified WDR11 as a gene involved in human puberty. We found six patients with a total of five different heterozygous WDR11 missense mutations, including three alterations (A435T, R448Q, and H690Q) in WD domains important for β propeller formation and protein-protein interaction. In addition, we discovered that WDR11 interacts with EMX1, a homeodomain transcription factor involved in the development of olfactory neurons, and that missense alterations reduce or abolish this interaction. Our findings suggest that impaired pubertal development in these patients results from a deficiency of productive WDR11 protein interaction.

Figure 1

Balanced Chromosome Translocation and FISH Mapping of the Chromosome 10 Breakpoint (A) Ideogram and composite chromosomes illustrating the balanced t(10;12)(q26.12;q13.11)dn (revised in this paper from the original karyotypic assessment, t(10;12)(q26.3;q13.1)dn) in the KS patient. (B) FISH mapping with BAC clone RP11-254K03, labeled with SpectrumGreen to metaphase spreads of the KS patient, resulted in hybridization to the normal chromosome 10, as well as the der(10) and der(12) chromosomes, indicating that the translocation breakpoint of chromosome 10 is located within the sequence of this BAC clone.

Figure 2

Genetic Mapping of the 10q26 Locus Involved in IHH and KS (A) The translocation breakpoint, depicted as a crocodile head, is located between SEC23IP and PPAPDC1A. Horizontal bars at 10q26.12 show BACs used for FISH mapping. The size and location of BACs are to scale. The blue bar depicts a BAC clone spanning the breakpoint. Four positional candidate genes located proximal and distal to the breakpoint are shown as arrows in a 2 Mb region. (B) Exon and intron structure of the 58 kb gene WDR11 (NM_018117.11). Locations of human missense mutations are identified in sporadic IHH and KS patients. Notable exons are shown to scale as blue rectangles and are numbered with exon size. The sizes of introns are not to scale. Mutation F1150L was identified in two independent sporadic IHH patients.

Figure 3

Sequences of the Translocation Breakpoints and Protein Sequence Alignment of WDR11 Orthologs (A) Genomic DNA sequence at the breakpoints from the normal and derivative chromosomes. The breakpoint on chromosome 10 is located between nucleotides 122,053,649 and 122,053,650, whereas on chromosome 12 it occurs between nucleotides 46,038,271 and 46,038,272 (UCSC Genome Browser NCBI hg18). The junction sequence reveals a 4 bp duplication at the breakpoint of the der(10) chromosome and a 1 bp deletion and a 57 bp insertion at the junction on the der(12) chromosome. (B) ClustalW multiple alignment of partial protein sequences of WDR11 orthologs. The positions of three residues affected by missense mutations of WDR11 are marked by arrows and red letters in the corresponding segments of the multiple alignment. The amino acid residues that differ from the sequence of the human WDR11 protein are indicated in blue, and the ninth WD domain is indicated under the panel. All three mutated residues are evolutionarily fully conserved in all 13 available WDR11 orthologs.

Figure 4

WDR11 Structural Model Indicating the Mutation Sites (A) Model spanning amino acids 70–739 of WDR11. The model was obtained by alignment to 1NR0 via ClustalW and MOE (Molecular Operating Environment [MOE 2004.03], Chemical Computing Group, Montreal, Quebec, Canada H3B 3X3). WDR11 forms a double propeller structure, in which the WD domains indicated in (B) form the main structural constituent. The two propeller axes are tilted with respect to one another, so only the propeller structure on the left is clearly visible in this representation. Colors indicate side chains of the four mutations within the modeled sequence region, as follows: WD domains predicted on the basis of the model (green), on the basis of SMART (pink), or both (cyan). The sites of the mutations are indicated in orange. (B) Positions of five missense mutations in WDR11; WD domains are depicted as ovals. The WD domains predicted on the basis of the model and by SMART are depicted in green and pink, respectively. The relative sizes and locations of WD domains are to scale. WDR11 contains twelve WD domains, nine (second to tenth repeats) that are confirmed on the basis of direct comparison with the template structure of AIP1 and three additional repeats (first, 11^th, and 12^th) detected by sequence comparison outside the region of the structural model. Note that three mutations directly affect WD domains 6 and 9.

Figure 5

The WDR11 Interacts and Colocalizes with EMX1 In Vivo and In Vitro (A) EMX1 was identified in the yeast two-hybrid screen as a WDR11-interacting protein. The specific interactions between these two proteins were confirmed by streaking of transformed yeast cells onto synthetic drop-out plates lacking TL (Trp/Leu) or TLH (Trp/Leu/His). Yeast AH109 cells were transformed with empty vectors (pGBKT7 and pGAD), and plasmids encoding SV40 T antigen (pTD1) and p53 (pGBKT7-p53) were utilized as a negative and positive control, respectively. (B) Myc-WDR11 expression plasmids were transfected alone or along with HA-EMX1 into HeLa cells, the cell lysates were immunoprecipitated with anti-Myc antibody, and coprecipitated HA-EMX1 was detected via immunoblotting with anti-HA antibody. (C) In-vitro-translated Myc-EMX1 was subjected to GST pull-down analysis with GST (lane 2) or GST-WDR11 (lane 3). The bound proteins were detected via immunoblotting with anti-Myc antibody. (D) HA-EMX1 and GFP-WDR11 expression plasmids were transfected into U2OS cells, and then cells were treated with leptomycin B (LMB), an inhibitor of nuclear export. Fluorescence microscopy analysis helped determine localizations of HA-EMX1 and GFP-WDR11. Nuclei were stained with DAPI. (E) Wild-type Myc-WDR11 and its deletion mutants were synthesized in vitro and subjected to a GST pull-down assay with GST (center panel) or GST-WDR11 (right panel). The N, M, and C denote the WDR11 N terminus (amino acids 1–361), middle portion (amino acids 362–830), and C terminus (amino acids 831–1224), respectively. The positions of missense mutations found within the N terminus and central region of WDR11 in IHH patients are marked as m1 through m4 on the schematic diagrams of WDR11. (F) The wild-type and missense mutant WDR11 expression plasmids were transfected into HEK293 cells along with HA-EMX1 expression plasmids. The cell lysates were immunoprecipitated with anti-Myc antibody, and the association of EMX1 with wild-type WDR11 or missense mutants was determined via immunoblot analysis with anti-HA antibody.

Figure 6

Wdr11 Expression during Murine Development (A–E) DIG-labeled whole-mount in situ hybridization with a Wdr11 antisense probe at different embryonic stages. High expression levels are found in all structures of the developing brain as early as E10.5. Expression in the limbs is prominent at E12.5 and E13.5. Staining was also observed in both the hind and forelimb buds, but as limbs developed, it shifted toward the terminal phalanges. At E14.5 the olfactory bulb and the developing cortex show the highest expression levels. A magnification of the developing cortex and olfactory bulb is shown in (E). (F–H) Expression of Wdr11 in the adult brain. [35S]-UTP-labeled in situ hybridizations show prominent Wdr11 signals in the piriform cortex (F) as well as in the hippocampus and cerebellum (G). Note the higher signal intensity in the hypothalamic region within the dotted rectangle in (G). Single cells as well as clusters of neurons within the hypothalamic nuclei also showed Wdr11 expression in DIG-labeled cryosections (H). Signals were absent with the sense control. Abbreviations are as follows: tel, telencephalic vesicle; wh, wall of hindbrain; di, diencephalon; mes, mesencephalon; rho, rhombencephalon; sc, spinal cord; lb, limb bud; hl, hind limb; fl, fore limb; ob, olfactory bulb; pc, piriform cortex; gcl, granule cell layer of the cerebellum; hp, hippocampus; and hy, hypothalamus.

Figure 7

Expression of wdr11 in Developing Zebrafish Embryos At 24 hpf, wdr11 transcripts were broadly detected in forebrain, midbrain, and hindbrain. (A, B, and E) Lateral view; anterior is to the left. (C and D) Dorsal view. The expression domain of wdr11 in the brain partially overlapped with that of emx1 (B and D), a dorsal telencephalon marker. Abbreviations are as follows: dt, dorsal telencephalon; f, forebrain; m, midbrain; h, hindbrain; mhb, midbrain-hindbrain boundary; vt, ventral telencephalon; and ret, retina.