Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2006 Jan;78(1):28-37.
doi: 10.1086/498879. Epub 2005 Nov 16.

Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype

D B Sparrow  1 , G Chapman, M A Wouters, N V Whittock, S Ellard, D Fatkin, P D Turnpenny, K Kusumi, D Sillence, S L Dunwoodie
Affiliations
Comparative Study

Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype

D B Sparrow et al. Am J Hum Genet. 2006 Jan.

Abstract

The spondylocostal dysostoses (SCDs) are a heterogeneous group of vertebral malsegmentation disorders that arise during embryonic development by a disruption of somitogenesis. Previously, we had identified two genes that cause a subset of autosomal recessive forms of this disease: DLL3 (SCD1) and MESP2 (SCD2). These genes are important components of the Notch signaling pathway, which has multiple roles in development and disease. Here, we have used a candidate-gene approach to identify a mutation in a third Notch pathway gene, LUNATIC FRINGE (LFNG), in a family with autosomal recessive SCD. LFNG encodes a glycosyltransferase that modifies the Notch family of cell-surface receptors, a key step in the regulation of this signaling pathway. A missense mutation was identified in a highly conserved phenylalanine close to the active site of the enzyme. Functional analysis revealed that the mutant LFNG was not localized to the correct compartment of the cell, was unable to modulate Notch signaling in a cell-based assay, and was enzymatically inactive. This represents the first known mutation in the human LFNG gene and reinforces the hypothesis that proper regulation of the Notch signaling pathway is an absolute requirement for the correct patterning of the axial skeleton.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Radiograph (A) and T2-weighted coronal MRI images (B and C) in the vertebral plane of the proband. A, Severe vertebral segmentation anomalies throughout the vertebral column. B, Thoracic spine, showing vertebral centers with a fitted angular shape. C, Cervical and lumbar spine, showing similar segmentation anomalies.
Figure 2
Figure 2
Detection of the c.564C→A mutation. A, Electropherograms documenting the affection status of the proband and parents. B, Confirmation of the presence of the c.564C→A mutation by MseI RFLP in genomic DNA isolated from an unrelated control individual, the proband, and the proband’s mother and father.
Figure 3
Figure 3
Structural model of LFNG, showing the proximity of the mutated phenylalanine residue (orange) to the Mn2+ binding site (Mn2+ in purple). Directly interacting residues F196 and H198 are shown in cyan, and the nearby Mn2+-ligating residues D202 and D203 are in red. The position of the UDP-sugar donor group (green) and an acceptor sugar (yellow) are shown for reference. α-helices and β-sheets are colored in a gradient from dark blue (amino terminus) to red (carboxy terminus).
Figure 4
Figure 4
A, Relative expression of transfected wild-type and mutant Lfng proteins. Shown is western blot detection of Lfng in lysates from C2C12 cells transfected with HA-tagged wild-type, TTA F187L, CTG F187L, and D202A Lfng. A total of 50 μg of each lysate was run on a 4%–12% PAGE gel and was blotted. Lfng proteins were detected using a mouse anti-HA antibody. Western blot detection of β-actin was performed to control for loading. B–D, Immunofluorescence analysis of wild-type and mutant Lfng proteins in cultured cells. F187L Lfng does not localize to the Golgi. C2C12 myoblasts were transiently transfected with constructs encoding HA-tagged wild-type Lfng (B), D202A mutant Lfng (C), and F187L Lfng (D). In each case, the top panel shows a confocal image of immunofluorescence with a rabbit anti-HA antibody, and the middle panel with the Golgi-specific antibody (GM130); the bottom panel shows a merged image. The scale bar represents 25 μm.
Figure 5
Figure 5
Results showing that F187 Lfng is enzymatically inactive. A, Western blot using an Lfng-specific antibody on Lfng-Fc fusion protein that was affinity purified from conditioned medium (“secreted”) or whole-cell extract (“intracellular”) derived from transiently transfected HEK 293T cells. B, GlcNAc-transferase assays performed on Lfng that was affinity purified from whole-cell extract by use of pNp-fucose (4 mM) as an acceptor and UDP-[3H]GlcNAc as a donor. Assays were performed in duplicate, and error bars represent SDs of the mean.
Figure 6
Figure 6
Results showing that F187L Lfng does not modulate ligand-induced Notch signaling. A, Wild-type Lfng significantly potentiates activation of Notch1 by coculture with Dll1-expressing cells, whereas F187L and D202A mutants do not alter levels of Notch1 activation. Three asterisks (***) denote P<.001 for wild-type versus F187L Lfng. B, Wild-type Lfng significantly inhibits activation of Notch1 by coculture with Jagged1-expressing cells, whereas F187L and D202A mutants do not alter levels of Notch1 activation. Three asterisks (***) denote P<.001 for wild-type versus F187L Lfng. Notch1 cells were cotransfected with plasmids encoding wild-type Lfng, mutant Lfng, or CAT (as a negative control) and a CSL reporter. Transfected cells were cocultured with either control NIH3T3, Dll1, or Jagged1 cells and were assayed for luciferase activity. Fold activation of ligand-expressing cells over control NIH3T3 cells is expressed relative to that of CAT-transfected cocultures. Notch signaling was activated 4–10-fold in these experiments. Error bars represent SDs of four independent experiments.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

Web Resources

    1. dbSNP, http://www.ncbi.nlm.nih.gov/SNP/
    1. Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for SCD1 and SCD2)

References

    1. Aulehla A, Johnson RL (1999) Dynamic expression of lunatic fringe suggests a link between notch signaling and an autonomous cellular oscillator driving somite segmentation. Dev Biol 207:49–6110.1006/dbio.1998.9164 - DOI - PubMed
    1. Bonafé L, Giunta C, Gassner M, Steinmann B, Superti-Furga A (2003) A cluster of autosomal recessive spondylocostal dysostosis caused by three newly identified DLL3 mutations segregating in a small village. Clin Genet 64:28–3510.1034/j.1399-0004.2003.00085.x - DOI - PubMed
    1. Bruckner K, Perez L, Clausen H, Cohen S (2000) Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406:411–41510.1038/35019075 - DOI - PubMed
    1. Bulman MP, Kusumi K, Frayling TM, McKeown C, Garrett C, Lander ES, Krumlauf R, Hattersley AT, Ellard S, Turnpenny PD (2000) Mutations in the human Delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis. Nat Genet 24:438–44110.1038/74307 - DOI - PubMed
    1. Capdevila J, Izpisua Belmonte JC (2001) Patterning mechanisms controlling vertebrate limb development. Annu Rev Cell Dev Biol 17:87–13210.1146/annurev.cellbio.17.1.87 - DOI - PubMed

Publication types

MeSH terms

Substances