Mutations in a novel gene, NHS, cause the pleiotropic effects of Nance-Horan syndrome, including severe congenital cataract, dental anomalies, and mental retardation

Abstract

Nance-Horan syndrome (NHS) is an X-linked disorder characterized by congenital cataracts, dental anomalies, dysmorphic features, and, in some cases, mental retardation. NHS has been mapped to a 1.3-Mb interval on Xp22.13. We have confirmed the same localization in the original, extended Australian family with NHS and have identified protein-truncating mutations in a novel gene, which we have called "NHS," in five families. The NHS gene encompasses approximately 650 kb of genomic DNA, coding for a 1,630-amino acid putative nuclear protein. NHS orthologs were found in other vertebrates, but no sequence similarity to known genes was identified. The murine developmental expression profile of the NHS gene was studied using in situ hybridization and a mouse line containing a lacZ reporter-gene insertion in the Nhs locus. We found a complex pattern of temporally and spatially regulated expression, which, together with the pleiotropic features of NHS, suggests that this gene has key functions in the regulation of eye, tooth, brain, and craniofacial development.

Figure 1

Families studied. A, Clinical features in individuals with NHS from the current study. Family 1, V-5: dentition (A.A), and cataract (A.B); family 5, II-2: dentition (A.C), and facial features (A.D); family 4, II-1: dentition (A.E); and family 4, I-1: cataract (A.F). Dental anomalies in the cases illustrated are diastema and notched incisors, showing striking similarity in three unrelated individuals. B, Pedigrees of the families with NHS. Xp22.13 haplotypes are shown for family 1, with the common haplotype boxed and recombinations indicated with an “x.” Blackened boxes denote affected males, blackened circles denote females with confirmed features of NHS, and unblackened boxes and circles denote unaffected males and females, respectively. ? = not available for examination.

Figure 2

Localization and characterization of the NHS gene. A, Schematic map of the Xp22.13 region, indicating positions of flanking markers and positions of candidate genes excluded, together with the structure of the NHS gene. Exon 1b defines a new putative NHS transcription start site. Note that markers DXS1195 and DXS418 are intragenic, residing in introns 1 and 5 of the NHS gene, respectively. Blackened segments indicate the ORF. Sequence chromatograms and positions of five truncation mutations identified in families 1–5 are shown. Blackened boxes next to chromatograms indicate affected males, blackened circles indicate affected females, and unblackened boxes indicate unaffected controls. B, Partial ClustalW multiple-protein alignment of NHS orthologs. Protein sequence encoded by exons 1–4 (upper panel) and exon 5 (lower panel) are shown. Mutations are indicated by arrows, and the nuclear localization sequence NLS1 is underlined. Conserved amino acid residues are boxed. Family numbers correspond to those in fig. 1B. Exon/exon boundaries are indicated with arrowheads and the corresponding exon numbers. The mouse protein sequence was based on mouse genomic sequences (AL672082, AC097354, AL732391, and AC093447) and mouse partial mRNAs similar to Nhs (XM_142285 and XM_112126).

Figure 3

Expression analysis of the NHS gene. A, RT-PCR from human adult brain, fetal brain, retina, and lens tissues. RNA was reverse transcribed and amplified using primers from exons 6 and 8. RT-minus controls were negative for each tissue. B, Mouse northern blot (Seegene), containing 20 μg of total RNA per lane from mouse brain at various developmental stages. The probe was generated from the 3′ end of the mouse Nhs gene. C, Mouse Nhs is developmentally regulated and is expressed in the brain, eye, and tooth primordia. C.A, Midsagittal section from E16.5 mouse embryo, showing Nhs expression in the choroid plexus of the fourth ventricle. C.B, Parasagittal section of E14.5 mouse embryo, showing Nhs expression in the midbrain. C.C, Sagittal section from E16.5 mouse embryo, showing expression in lens cell bodies (arrow) and retina (arrowhead). C.D, Sagittal section from E16.5 mouse embryo, showing Nhs expression in the primordium of upper right incisor tooth (arrow) and the whisker follicles (arrowhead). C.E, Detail of panel D, showing Nhs expression in the primordium of upper right incisor tooth (arrow). C.F, Parasagittal section from E16.5 mouse embryo, showing Nhs expression in the primordia of the upper and lower right molar teeth (arrows). C.G, Midsagittal section from E14.5 mouse embryo, showing Nhs expression in the right ventricle of the heart (arrow). C.H, Parasagittal section from E14.5 mouse embryo, showing NHS expression in olfactory epithelium (arrows). The Nhs4 probe was used in panels A and G, and the Nhs5 probe was used in panels B, C, D, E, F, and H.

Figure 4

Expression analysis of the Nhs locus, using the lacZ reporter gene. Shown is β-galactosidase staining of a parasagittal section of an E10.5 embryo (A), a whole-mount E11.5 embryo (B), a whole-mount E12.5 embryo (C), and a coronal section of an E12.5 eye (D); radioactive in situ hybridization of a coronal section of E12.5 eye using the Nhs5 probe (E) and bright field of E (F); β-galactosidase staining of whole-mount PN0 brain (dorsal aspect) (G), whole-mount PN0 brain (ventral aspect) (H), adult brain (slice at frontal level) (I), and adult brain (slice at parietal level) (J). AER = apical ectodermal ridge; AN = amygdaloid nuclei; ANP = amygdaloid nuclei primordium; Co = cortex; dMan = dorsal mandibular process; He = heart; Hi = hippocampus; Hy = hypothalamus; Le = lens; LCB = lens cell bodies; LV = lens vesicle; Med = medulla; Mes = mesencephalon; NR = neural retina; OB = olfactory bulb; OE = olfactory epithelium lining the olfactory pit; ON = optic nerve; OT = olfactory tubercle; Po = pons; PR = pigmented retina; SN = septal nuclei; TG = trigeminal ganglion; vMax = ventral maxillary process; vMes = ventral mesencephalon; vNT = ventral neural tube; vSC = ventral spinal cord.