HOXB1 founder mutation in humans recapitulates the phenotype of Hoxb1-/- mice

Abstract

Members of the highly conserved homeobox (HOX) gene family encode transcription factors that confer cellular and tissue identities along the antero-posterior axis of mice and humans. We have identified a founder homozygous missense mutation in HOXB1 in two families from a conservative German American population. The resulting phenotype includes bilateral facial palsy, hearing loss, and strabismus and correlates extensively with the previously reported Hoxb1(-/-) mouse phenotype. The missense variant is predicted to result in the substitution of a cysteine for an arginine at amino acid residue 207 (Arg207Cys), which corresponds to the highly conserved Arg5 of the homeodomain. Arg5 interacts with thymine in the minor groove of DNA through hydrogen bonding and electrostatic attraction. Molecular modeling and an in vitro DNA-protein binding assay predict that the mutation would disrupt these interactions, destabilize the HOXB1:PBX1:DNA complex, and alter HOXB1 transcriptional activity.

Figure 1

Clinical and Neuroimaging Phenotype of Patients Harboring the Homozygous HOXB1 c.619C>T mutation (A) Photographs of affected brothers from family A show a “masked facies” appearance secondary to bilateral facial weakness (patients VI-1 and VI-3 in Figure 2A). Dysmorphic features including midface retrusion, an upturned nasal tip, and low-set and posteriorly rotated ears are noted (Figure S1). Residual postoperative right concomitant esotropia is seen in the younger brother (VI-3), pictured on the right. (B) Gaze positions of the younger affected brother in family A (VI-3) reveal full ocular motility with right esotropia, thus ruling out Moebius syndrome. Primary gaze (center), right gaze (left), left gaze (right), upgaze (top), and downgaze (bottom). (C–E) Axial 3D FIESTA (fast imaging employing steady state acquisition) MR imaging of the older brother in family A (VI-1) at 8 months of age. (C) Image is taken through the posterior fossa at the level of the internal auditory meati. The vestibulocochlear nerves (arrows) are demonstrated exiting the pontomedullary junction bilaterally and traversing the cerebellopontine angle cisterns. The facial nerves are not visualized and would ordinarily be expected to travel ventrally and parallel to the VIIIth cranial nerves. (D) Image is a more caudal view demonstrating the right vestibulocochlear nerve (arrow) entering the internal auditory meatus; there is no evidence of a facial nerve in its expected location more ventrally. The vestibulocochlear nerve (arrow) is demonstrated bifurcating into the cochlear and inferior vestibular nerves at a level caudal to the expected location of the VIIth cranial nerve. (E) Image is a more caudal view that reveals subtle bilateral abnormal tapering of the basal turn of the cochlea (short arrow). (F) Photographs of affected siblings from family B were taken of the sister as a young child and the brother as an adult (patients II-2 and II-4 in Figure 2B). Both have masked facies and bilateral facial weakness, as exhibited in the brother's attempt to smile. The brother has a postoperative right exotropia that is probably secondary to overcorrection. Both siblings also have midface retrusion, an upturned nasal tip, and micrognathia. The brother has low-set ears.

Figure 2

Family Structure, Genotyping, and Haplotype Analysis (A and B) Schematic representations of pedigree structures, founder haplotype, and mutation status of families A (A) and B (B). Squares denote males, circles denote females, and shaded symbols denote affected individuals. Individuals were genotyped for 20 tagging SNPs on chromosome 17q21: centromere- rs199457, rs3760377, rs2002537,rs1515752, rs2292699, rs4794047, rs6503934, rs3897986, rs1533057, rs1509635, rs17697950, rs6504280, rs1553748, rs11869101, rs925284, rs11079824, rs10853100, rs11079828, rs8073963, and rs2229302- telomere. The mutation occurs between rs10853100 and rs11079828. A parsimony approach was used for phasing haplotypes. Representative haplotypes consisting of five SNPs that are informative in these two families are shown (bolded above). The disease-bearing haplotype is boxed, and this haplotype is shared on chromosomes with the HOXB1 c.619C>T mutation in both families. (C) Chromatograms of an unaffected individual (top), a mutation carrier (middle), and an affected individual (bottom). There is a homozygous C>T substitution at residue 207 in the affected individual in the position indicated by the red arrow. The wild-type and altered amino acid residues are noted below each sequence.

Figure 3

HOXB1 Protein Structure, Conservation, and Molecular Modeling (A) A two-dimensional schematic representation of HOXB1 highlights the homeodomain (in red brackets), containing 3 α helices and an N-terminal arm. The Arg207Cys amino acid substitution, indicated by the red arrow head, falls in the N-terminal arm of the homeodomain. The H indicates the position of an Antp-type hexapeptide motif. (B) HOXB1 Arg207 corresponds to the Arg5 residue of the HOXB1 homeodomain. This residue is highly conserved phylogenetically (top) and among other human HOX proteins (middle). Arg5 is also conserved in other homeodomain-containing proteins (bottom), for which mutations altering Arg5 cause human disease. (C–E) Protein modeling of the Arg207Cys mutation in the HOXB1:PBX1:DNA ternary complex (PDB:1B72). (C and D) Wild-type Arg5 residue (green R5) of the HOXB1 homeodomain interacts with the O2 atoms of the T11 base of the DNA with three hydrogen bonds at 2.6Å, 3.7Å, and 4.8Å. (E) The sulfur atom of mutant Arg5Cys residue (red C5) of the HOXB1 homeodomain would interact with the oxygen atom of T11 base of the DNA via one hydrogen bond at 6.0Å.

Figure 4

Transactivation of Human HOXB1 Wild-Type and Mutant Proteins (A) The relative activity (firefly luciferase/Renilla luciferase) for the wild-type and the c.619C>T mutant was measured with varying amounts of plasmid (25, 50, 75, and 100 ng). The experiment was completed in triplicate, and the luciferase activities were measured 48 hr post-transfection (the wild-type is delineated with a dashed line, and the mutant is delineated with a solid line). Means ± SDs are shown, and all p values are less than 0.05. Transfections were carried out with 200 ng of pAdML-ARE, 200 ng of pSG-PBX1A, and 50 ng of pRL null with either pSG-HOXB1 wild-type or mutant plasmids via Lipofectamine LTX (Invitrogen) in HEK293T cells grown in Dulbecco's Modified Eagle Medium supplemented with 10% fetal calf serum. Experiments were performed in a 24-well plate, 12–16 hr after 100,000 cells were plated in 0.5 ml of medium per well. Simultaneously, negative control experiments including the absence of HOXB1 vector at transfection or the replacement of pML-HOXB1-ARE with pAdML were conducted and gave expected lower luciferase activity ratios (data not shown). (B) The percentage of wild-type activity for the Arg207Cys HOXB1 mutant construct is shown.