Distinct effects of allelic NFIX mutations on nonsense-mediated mRNA decay engender either a Sotos-like or a Marshall-Smith syndrome

Abstract

By using a combination of array comparative genomic hybridization and a candidate gene approach, we identified nuclear factor I/X (NFIX) deletions or nonsense mutation in three sporadic cases of a Sotos-like overgrowth syndrome with advanced bone age, macrocephaly, developmental delay, scoliosis, and unusual facies. Unlike the aforementioned human syndrome, Nfix-deficient mice are unable to gain weight and die in the first 3 postnatal weeks, while they also present with a spinal deformation and decreased bone mineralization. These features prompted us to consider NFIX as a candidate gene for Marshall-Smith syndrome (MSS), a severe malformation syndrome characterized by failure to thrive, respiratory insufficiency, accelerated osseous maturation, kyphoscoliosis, osteopenia, and unusual facies. Distinct frameshift and splice NFIX mutations that escaped nonsense-mediated mRNA decay (NMD) were identified in nine MSS subjects. NFIX belongs to the Nuclear factor one (NFI) family of transcription factors, but its specific function is presently unknown. We demonstrate that NFIX is normally expressed prenatally during human brain development and skeletogenesis. These findings demonstrate that allelic NFIX mutations trigger distinct phenotypes, depending specifically on their impact on NMD.

Figure 1

Genetic Analyses of Patients with Syndromic Overgrowth (A) The figure shows the Ensembl display of the 19p13.2p13.3 region. Position of nonoverlapping NFIX deletions detected in patients A and B are represented by squares. For patient A, we found the proximal and distal breakpoints at 12 898 751 and 13 006 539 bp, respectively. For patient B, we found the proximal and distal breakpoints at 13 022 358 bp and 13 126 508 bp, respectively. (B) The diagram shows the different mutations identified in NFIX genomic DNA. Open boxes represent untranslated regions and filled boxes represent coding regions. Mutation in green corresponds to the Sotos-like case while those in red indicate mutations found in MSS cases. (C) Representative sequence traces of exon 3 in genomic DNA (top) or cDNA (bottom) of patient C (c.568C>T). (D) Representative sequence traces of exon 8 sequences from genomic DNA (top) or cDNA (bottom) of patient F (c.1243 delG).

Figure 2

Front Views of the Three Overgrowth Syndrome Patients Note the high forehead, long narrow face (patients A and C), and slender habitus.

Figure 3

Pictures and Hand and Foot X-Rays of Patients with Marshall-Smith Syndrome Facial features of the nine patients with Marshall-Smith syndrome (A–I) and hand X-rays at 1 year (J) and 4 years (K) of age and foot X-rays at 1 year of age (L). Note the high forehead, proptosis, underdeveloped midface, the advanced carpal and tarsal ossification, and the abnormal wide, bullet-shaped phalanges.

Figure 4

Pattern of NFIX Expression in Early Human Development and Fetal Epiphyseal Growth Plate (A–G) Adjacent sections of human embryos and fetuses treated with antisense (A–C, E–G) or with control sense (D) riboprobes. (A) Carnegie stage 17 (CS17) human embryo section. (B–D) CS19 human embryo sections. (E) Frontal section of a fetal (22WG) brain. (F and G) Sagittal section of a fetal (14WG) hand. The hybridization with control sense probe does not give any signal (D). Note the expression in the neuroepithelium of the prosencephalon, the mesencephalon, the rhombencephalon, and the spinal cord as well as in the cranial and dorsal root ganglia. Abbreviations: Ah, Ammon's horn; c, cerebral cortex; co, cochlea; dg, dentate gyrus; di, diencephalon; drg, dorsal root ganglia; fv, fourth ventricule; h, heart; hu, humerus; ie, inner ear; la, lateral ventricule; li, liver; lu, lung; M, mesencephalon; me, mesonephros; nt, neuroepithelium of the neural tube; oe, olfactory epithelium; op, optic stalk; ot, otic vesicle; p, pons; pa, pharingial arch; sca, scapula; st, stomach; T, Telencephalon; th, thalamus; V, trigeminal ganglia; ve, cartilage primordia of vertebrae. (H–M) NFIX immunohistochemostry performed in 1-week-old mice (H), 2-week-old mice (I), 3-week-old mice (J), 4-week-old mice (K), 5-week-old mice (L), and 20 weeks of gestation human fetus (M). Note the restricted expression in the prehypertrophic chondrocytes and the expression in bone. Abbreviations: of, ossification front, hy, hypertrophic zone; PHy, prehypertrophic zone; Pro, proliferative zone.