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Blood, Vol. 92 No. 8 (October 15), 1998:
pp. 2987-2989
CORRESPONDENCE
FGFR3 Gene Mutations Associated With Human Skeletal
Disorders Occur Rarely in Multiple Myeloma
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LETTER |
To the Editor:
Fibroblast growth factor receptor 3 (FGFR3) is one of four distinct
tyrosine-kinase receptors (FGFR1-4) that are capable of binding a
repertoire of at least nine related mitogenic fibroblast growth factors
(FGFs). FGFRs encode proteins that all contain three
glycosylated extracellular Ig-like domains, a transmembrane domain
(TM), and a split cytoplasmic tyrosine-kinase domain. Point mutations
in distinct domains of the FGFR3 gene are associated with
autosomal dominant human skeletal disorders, such as achondroplasia, thanatophoric dysplasia types I and II, and
hypochondroplasia.1,2 Recent reports indicate that the
point mutations associated with these disorders produce constitutively
activated FGFR3, which shows autophosphorylation in the absence of
ligand and is no longer regulated by FGF binding.3-6
We and others have recently provided the first evidence of
FGFR3 gene involvement in human cancer.7,8 In
particular, the FGFR3 gene located at 4p16.3 is translocated to
chromosome 14q32 as a result of a novel and karyotypically undetectable
t(4;14)(p16.3;q32) chromosomal translocation in multiple myeloma (MM),
a malignant proliferation of plasma cells. Molecular studies have shown
this lesion in five MM-derived cell lines and in four primary tumors. Although the breakpoints on 4p16.3 are located approximately 50 to 120 kb centromeric to FGFR3, the gene is overexpressed in these cases, but absent or barely detectable in cell lines without the translocation. Interestingly, FGFR3 gene mutations associated with distinct human skeletal disorders2 have also been
identified in some MM tumors carrying the t(4;14)(p16.3;q32): in
particular, the Y373C mutation in the KMS-11 cell line,7,8
the K650E mutation in the OPM2 cell line,7 and the K650M
mutation in a primary MM tumor.7
These findings prompted us to look for FGFR3 mutations known to
be associated with skeletal disorders in a representative panel of MM,
including 80 primary cases (60 patients at first diagnosis, 12 at
relapse, and 8 affected by plasma cell leukemia) and 10 MM-derived cell
lines (including the KMS-11 and OPM2 cell lines). The analysis was
performed by means of the polymerase chain reaction-single-strand
conformation polymorphism (PCR-SSCP) direct sequencing of genomic
DNA. We amplified five distinct genomic FGFR3 fragments containing codons affected by mutations: codon 248, the entire TM domain (codons 371, 373, 375, and 380), codon 540, codon 650, and codon 807 (Fig 1). The
mutations at codon 650 were also investigated by means of a restriction
enzyme analysis of the PCR-amplified fragment using Mbo II and
Bbs I enzymes, as previously described.9 We
detected allelic variations of the FGFR3 gene only in the
fragment specific for the TM domain. An abnormal fragment with the same
pattern of migration was observed in 2 cases (the LP-1 cell line and a
primary tumor; Fig 2); in both cases, a
novel single basepair mutation involving codon 384 in the form of a T
to C transition (TTC-CTC) led to a conservative Phe  Leu amino acid
substitution (data not shown). Interestingly, this mutation abrogates a
Mbo II restriction site and creates a new Mnl I site
that allows restriction enzyme analysis of the PCR-amplified fragment.
The apparently similar intensity of the normal and mutated bands in
both cases, as well as the detection of the mutation in 2 of 100 normal
individuals by means of restriction enzyme analysis, suggest that it
may represent a rare genetic polymorphism. Finally, the FGFR3
gene was apparently not expressed in the LP-1 cell line; it remains to
be seen whether this particular variant may affect FGFR3 biological
activity.
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| Fig 1.
Schematic representation of the primers from the human
FGFR3 gene used in the study. The FGFR3 exons are
indicated by white boxes, and the introns are indicated by lines. The
3 untranslated region is indicated by the dashed box. The approximate
locations of the primers, the length of the amplified fragments, and
the approximate positions of codons 248, 540, 650, and 807 are
indicated. The nucleotide sequence of FGFR3 cDNA and the
intron-exon organization of the gene have been previously
reported.12,13 The sequences of the primers are as follows:
248F (intron 6), 5-CCTGAGCGTCATCTGCC-3, and 248R (exon 7),
5-CCATTGCATCCCACACGG-3; TD5 (exon 10), 5-AGGAGCTGGTGGAGGCTGA-3, and
TD3 (exon 10), 5-GGAGATCTTGTGCACGGTGG-314; 540F (exon
13), 5-ACTGACAAGGACCTGTCGGAC-3, and 540R (exon 13),
5-GCCCTGCGTGCAGGCGCC-3; 650F (exon 15), 5-GCATCCACAGGGACCTGG-3, and
650R (intron 15), 5-AGGCGGTGTTGGCGCCAG-3; 14S (exon 15),
5-GTGCACAACCTCGACTAC-3 (this primer was used with 650R to obtain a
DNA fragment suitable for the restriction enzyme analysis of codon
650); 807F (exon 19), 5-CCTGTCGGCGCCTTTCGAGCAGTAC-3, and 807R (exon
19), 5-CACCAGCAGCAGGGTGGGCTGCTAG-3.15
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| Fig 2.
PCR-SSCP analysis of the FGFR3 gene. N, normal
control; migrating fragments different from the normal control are
indicated by arrows.
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Although no specific genetic lesions have been found to be associated
with MM (unlike other types of lymphoid neoplasms), cytogenetic and
more recent molecular analyses suggest that chromosomal translocations
involving the Ig locus on chromosome 14q32 may play an important role
in gene deregulation.7,8 In this context, the recent
identification of the t(4;14)(p16.3;q32) in MM, associated with an
apparent deregulation of the FGFR3 gene, may provide some insights into the pathogenesis of this neoplasia. Although more work is
needed to assess the role and frequency of the t(4;14) in MM, it can be
suggested that deregulation of FGFR3 gene expression may lead
to a constitutive oncogenic signal for the growth and/or survival of malignant plasma cells. This possibility is supported by
the evidence that the bone marrow environment and, in particular, the
stromal cells with which the plasma cells interact10
are able to produce FGFs.11 The FGFR3 mutations
reported in MM probably represent somatic events, suggesting that
FGFR3 gene may be deregulated by different mechanisms. However,
we were unable to detect FGFR3 mutations associated with
skeletal disorders in our series of samples, except in the cell lines
previously reported.7,8 This finding suggests that such
mutations represent rare events in MM and support the hypothesis that
they may occur after the translocation and deregulation of the
FGFR3 gene, thus contributing to tumor progression by means of
ligand-independent activation.
Nicola Stefano Fracchiolla
Stefano Luminari
Luca Baldini
Luigia Lombardi
Anna Teresa Maiolo
Antonino Neri
Servizio di Ematologia
Istituto di
Scienze Mediche
Università di Milano
Ospedale
Maggiore
IRCCS
Milan, Italy
| |
ACKNOWLEDGMENT |
We are grateful to Dr T. Otsuki, Dr F. Malavasi, and Dr A. Solomon for
providing us with the some of the MM-derived cell lines (KMM1, KMS-11,
KMS-12, LP-1, and UTMC-2) used in this study and to G Ciceri for
technical assistance. The cell lines U266, Sultan, ARH-77, and RPMI
8226 were obtained from ATCC and the OPM2 cell line was obtained from
DSMZ. This work was supported by a grant from the Associazione Italiana
Ricerca sul Cancro (AIRC) to A.N. and a grant "Ricerca Corrente
1994" from the Ministero Italiano della Sanità to Ospedale
Maggiore IRCCS.
| |
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