Heterozygous mutations of OTX2 cause severe ocular malformations

Abstract

Major malformations of the human eye, including microphthalmia and anophthalmia, are examples of phenotypes that recur in families yet often show no clear Mendelian inheritance pattern. Defining loci by mapping is therefore rarely feasible. Using a candidate-gene approach, we have identified heterozygous coding-region changes in the homeobox gene OTX2 in eight families with ocular malformations. The expression pattern of OTX2 in human embryos is consistent with the eye phenotypes observed in the patients, which range from bilateral anophthalmia to retinal defects resembling Leber congenital amaurosis and pigmentary retinopathy. Magnetic resonance imaging scans revealed defects of the optic nerve, optic chiasm, and, in some cases, brain. In two families, the mutations appear to have occurred de novo in severely affected offspring, and, in two other families, the mutations have been inherited from a gonosomal mosaic parent. Data from these four families support a simple model in which OTX2 heterozygous loss-of-function mutations cause ocular malformations. Four additional families display complex inheritance patterns, suggesting that OTX2 mutations alone may not lead to consistent phenotypes. The high incidence of mosaicism and the reduced penetrance have implications for genetic counseling.

Figure 1

OTX2 mutation analysis in eight families with congenital eye malformations. Top, Schematic diagram of the human OTX2 cDNA, showing the homeodomain (“HD”) (red rectangle); SIWSPA motif (dark blue bar); SGQFTP motif (orange bar); and tandemly repeated OTX-tail motif (dark blue triangles). NTD = N-terminal domain; CTD = C-terminal domain. Horizontal arrows show the extent of the three exons. Vertical arrowheads show the position of each mutation, with the corresponding family number. Bottom, Pedigrees with representative sequence traces. Genetic analysis was performed for all individuals except those with dashed outlines. Blackened symbol = affected; unblackened symbol = unaffected; dashed outline = individual not available for analysis; blackened dot in unblackened symbol = unaffected mutation carrier; unblackened dot in blackened symbol = affected individual without mutation; m = mosaic. Numbers to the left of each sequence trace indicate generation and individual; patient numbers are also shown (in parentheses). In family 3, individuals II-1 and II-2 are second cousins. For family 4, “N” is a normal control sequence for comparison. Arrowheads below the traces for individuals II-2 (family 3) and I-2 (family 4) indicate a low-level presence of the mutant allele.

Figure 2

Facial views of eight patients with OTX2 coding-region sequence changes: patient 1 (A), patient 3A (B), patient 4A (C), patient 4B (D), patient 5 (E), patient 6 (F), patient 7 (G), and patient 8 (H). Patients 5 and 7 are wearing prostheses in both eyes. Patient 8 is wearing a prosthesis in the left eye.

Figure 3

Ocular findings for patient 2 at age 6 wk. The right eye (A) and left eye (B) are shown, both with persistent pupillary membrane and tunica vasculosa lentis and a clear lens. Ultrasound scans of the right (C) and left (D) orbits show small eyes. In panel C, no optic nerve is visible. In panel D, the thin cord extending posteriorly from the globe (arrowhead) may be the optic-nerve sheath. The left fundus (E) has a white elevated area with a pigmented surrounding area and grayish tissue extending a short distance into the posterior segment. This may be a hyaloid remnant. No retinal vessels are visible. The fundus is hypopigmented, with a granular appearance and a few choroidal vessels. F, In electrophysiological tests, patient 2 (left) gave a small (<2 mV) but consistent response to flash “a” and “b” waves (control >10 mV). Visual evoked potential (“VEP”) showed no consistent response to flash. Results from an age-matched control are shown (right).

Figure 4

Ocular findings in family 4. Patient 4B at age 28 years: right fundus (A) and left fundus (B), both with pale optic disks, atrophic maculae, and thin retinal vessels (arrowheads); right iris (C) and left iris (D). The left iris has peripheral anterior synechiae (arrow). Patient 4C (mosaic mother of patients 4A and 4B) at age 51 years: right fundus (E) and left fundus (F), showing retinopathy with accumulated pigment clumps (arrowheads).

Figure 5

Coronal MRIs from patient 5 (T1-weighted image [A]), a normal control (T1-weighted image [B]), and patient 6 (T2-weighted image [C]). The left side of each image is the right side of the patient. In panels A and C, the hippocampi are circled. Panel B is labeled to show the normal hippocampal and parahippocampal structures: 1 = body of the hippocampus; 2 = subiculum; 3 = ambient cistern; 4 = parahippocampal gyrus; 5 = collateral sulcus; 6 = fusiform gyrus; 7 = lateral occipitotemporal sulcus. The hippocampi and parahippocampal structures are normal in panel A but are abnormal in panel C. Despite the different image parameters (T2 vs. T1 control), in panel C the hippocampi are more vertically aligned and are more globular than ovoid. Instead of being tucked laterally into the temporal horns of the lateral ventricles, the medial displacement and incomplete rotation of the hippocampi generate a cerebrospinal fluid–filled extension of the temporal horns of the lateral ventricle (arrow).

Figure 6

Expression pattern of the human OTX2 gene in the developing head (A–E) and eye (F–H). Panels show representative results of nonradioactive in situ hybridization with a human OTX2 probe. All panels (apart from panel C, which shows a sense control) show antisense hybridization: CS14 sagittal (A); CS16 coronal (B and C); CS18 (D); CS22 sagittal (E); CS16 5× magnification of panel B (F); CS17 coronal (G); CS19 transverse (H). Strong expression is seen in the RPE (panels B, D, F, G, and H), and weaker expression is seen in the neural retina. Pigment formation has not yet begun at this stage; cp = choroid plexus; di = diencephalic vesicle; dt = dorsal thalamus; ey = eye; l = lens; lt = lamina terminalis; me = medial eminence; mes = mesencephalic vesicle; mhb = midbrain/hindbrain boundary; mt = metencephalic vesicle; my = myelencephalic vesicle; np = nasal pit; nr = neural retina; os = optic stalk; rh = rhombencephalic vesicle; rpe = retinal pigment epithelium; tel = telencephalic vesicle; vt = ventral thalamus.