MUTATION IN BRIEF
HUMAN MUTATION Mutation in Brief #487 (2002) Online
© 2002 WILEY-LISS, INC.
Received 19 September 2001; accepted 21 December 2001.
Identification of a Novel NOG Gene Mutation (P35S)
in an Italian Family with Symphalangism
M. Mangino1, E. Flex1, M.C. Digilio2, A. Giannotti2, and B. Dallapiccola1
1C.S.S. Mendel Institute and University La Sapienza, Rome, Italy; 2Division of Medical Genetics Bambino Gesù
Hospital, Rome, Italy.
*Correspondence to: Prof. Bruno Dallapiccola, C.S.S. Mendel Institute Viale R. Margherita 261, 00198 Rome,
Italy; Tel.: +390644160503; Fax: +390644160548; E-mail: firstname.lastname@example.org
Communicated by Richard G. H. Cotton
Symphalangism (SYM or SYM1) is an autosomal dominant disorder characterized by
multiple joint fusions. The disease is caused by mutations of the NOG gene, that maps to
chromosome 17q22. So far, only six independent NOG mutations have been identified. We
have analysed an Italian family in which father and son had bilateral symphalangism and
detected a novel NOG mutation (P35S), originated in the father from a c.914C>T transition.
A different mutation in the same codon (P35R) has been previously described. Comparison
between different noggin gene hortologs shows that codon 35 is conserved. Therefore, this
codon should play an important role in NOG gene function. This is the first mutation
described for NOG after the initial report of NOG mutations being causative of SYM. © 2002
KEY WORDS: Noggin; NOG; Symphalangism; SYM1; joint malformation
Symphalangism (SYM1; OMIM 185800) is an autosomal dominant disorder characterized by early onset
progressive ankylosis of the proximal interphalangeal joints, carpal and tarsal bone fusion (Cushing, 1916), and
conductive hearing loss (Vesell, 1960). In 1995, the SYM1 locus was mapped to chromosome 17q21-22
(Polymeropoulos et al., 1995). Subsequently, Gong et al. (1999) identified five familial noggin gene (NOG; OMIM
602991) mutations segregating with proximal symphalangism, and a de novo mutation in a patient with unaffected
parents. These authors also described a NOG mutation in a family with multiple synostoses syndrome (SYNS1;
OMIM 186500), demonstrating that SYM1 and SYNS1 are allelic disorders. All the seven NOG mutations
described so far alter evolutionarily conserved amino acid residues (Gong et al., 1999). Here we report a novel
mutation in the NOG gene segregating in two generations of an Italian family with symphalangism.
MATERIALS AND METHODS
We analysed an Italian family (Fig. 1) in which father and son had bilateral symphalangism of fingers 2-5 and
toes 3-4, short first metacarpals, distal hand phalangeal hypoplasia, and mild conductive hearing loss. The father
had also thoraco-lumbar scoliosis. Facial appearance was unremarkable in both patients.
2 Mangino et al.
Blood samples were obtained from the family members and DNA was extracted using standard protocols.
Markers D17S1868-D17S787-D17S944 from the ABI-PRISM Linkage Mapping Set Version II were genotyped.
PCR was carried out under standard conditions, and amplified products were loaded in an ABI3100 Genetic
Analyser. Allele size was defined using the Genescan 3.5 software. Chromosome 17 haplotypes were derived by
minimizing recombination events.
Primers for mutation analysis were designed on the NOG gene sequence (GenBank no. U31202) and are
available on request. For exon amplification, genomic DNA (100ng) was denatured at 95°C for 5', mixed with 1x
buffer, 1.5mM MgCl2, 0.5mM primers, 200mM dNTP, 1U Taq polymerase in a final volume of 50 µl, and cycled
30x at 94°C for 30’’, 60°C for 30’’, 72°C for 30’’ and finally incubated at 72°C for 5 minutes. Fluorescent DNA
sequencing was performed with a 310 Genetic Analyser, using the PRISM Dye Terminator kit. All potential DNA
sequence changes were verified by DNA sequence analysis on both strands from at least two independent PCR
products. Additionally, DNA sample from 100 normal alleles were screened to confirm the mutation.
The PCR products (412 bp) were digested with MspI for 3 hours at 37°C. Electrophoresis was performed in
2% MS-8 Hispanagar agarose gels, and bands visualized by ethidium bromide staining.
Protein prediction analysis
Computer modelling of the mutant protein predicted structure was carried out with the PHDsec software (Rost
& Sander, 1994) available on line at http://cubic.bioc.columbia.edu/predictprotein/
RESULTS AND DISCUSSION
Analysis of the NOG gene coding region revealed a C>T substitution at nucleotide 914 in subjects II:1 and
III:1. Restriction analysis confirmed this mutation only in the affected subjects II:1 and III:1, while unaffected
parents I:1 and I:2 showed the wild-type pattern (Fig. 1). Paternity testing was carried out by analyzing a set of
chromosome 17 markers. Haplotype analysis confirmed that II:1 had inherited a chromosome 17 from each of his
parents (Fig. 2).
The c.914C>T mutation results in a non-conservative amino acid change of a proline to a serine at codon 35. A
different amino acid change (P35R) has also been described in this evolutionarily conserved codon (Gong et al.,
1999). The identification of two independent point mutations suggests that this could be a mutation hot spot. The
substitution of proline for serine is likely to induce significant topological changes, as predicted by the secondary
structural analysis. The beta sheet formed between the T10 and A13 residues in the wild-type protein is abolished
by the mutation, which also leads to an extension of the helix between T10 and R21 residues (Fig. 3). This
conformational change might inhibit NOG dimer formation or its binding to the subset of the TGFb-FMs with
which it normally interacts (Gong et al., 1999).
Characterization of new NOG mutations and in vivo expression studies using X. laevis and D. melanogaster
oocytes may lead to understand the precise effects of these mutations.
Novel NOG Mutation in SYM1
Figure 2. Chromosome 17q haplotype analysis. Filled symbols show the affected individuals. Black bars
indicate the at risk aplotype.
Figure 1. Confirmation of the P35S mutation in genomic DNA. The mutation abolishes an MspI
restriction site. Affected individuals show a 184 bp fragment. Subjects I:1 and I:2 show a wild-type
pattern. M: Marker; ND: Non-digested fragment.
4 Mangino et al.
PHD sec | EEEEHHHHHHHEE EEEEEE |
SUB sec |LLLLLLL...............LLLLL.EEEE.LLLLLLL|
PHD sec | HHHHHHHHHHH EEEEEE |
SUB sec |LLLLLLLL..HHHHHHHH......LLL.EEEE...LLLLL|
Figure 3. Secondary-structure prediction for the wild-type noggin sequence and the P35S
mutation using the PHDsec algorithm. The mutated residues are in bold face and underlined.
E=extended sheet (beta sheet); H = helix; L = loop. The prediction is meaningful for all
residues with an expected average correlation > 0.69.
Cushing H. 1916. Hereditary anchylosis of proximal phalangeal joints (symphalangism). Genetics 1:90-106.
Gong Y, Krakow D, Marcelino J, Wilkin D, Chitayat D, Babul-Hirji R, Hudgins L, Cremers CW, Cremers FP, Brunner HG,
Reinker K, Rimoin DL, Cohn DH, Goodman FR, Reardon W, Patton M, Francomano CA, Warman ML. 1999.
Heterozygous mutations in the gene encoding noggin affect human joint morphogenesis. Nat Genet 21:302-304.
Polymeropoulos MH, Poush J, Rubenstein JR, Francomano CA. 1995. Localization of the gene (SYM1) for proximal
symphalangism to human chromosome 17q21-q22. Genomics 27:225-229.
Rost B, Sander C. 1994. Combining evolutionary information and neural networks to predict protein secondary structure.
Vesell ES. 1960. Symphalangism, strabismus and hearing loss in mother and daughter. N. Engl. J. Med. 263:839-842.