Animal model for fibroblast growth factor receptor...

Details Animal model for fibroblast growth factor receptor... Animal model for fibroblast growth factor receptor...

1999-08-26

2001-07-24

Hauda, Karen M. (Department: 1632)

Multicellular living organisms and unmodified parts thereof and

Nonhuman animal

Transgenic nonhuman animal

C800S008000, C800S009000

Reexamination Certificate

active

06265632

ABSTRACT:

FIELD AND BACKGROUND OF THE INVENTION
The present invention relates, in general, to an animal model for chondrodysplasia, and more particularly, to a transgenic mouse model for achondroplasia in which a fibroblast growth factor receptor 3 gene including a G to A point mutation changing Gly to Arg in codon 380 thereof (numbered according to the human sequence) is introduced into the mouse genome.
Achondroplasia is the most common genetic form of osteochondrodysplasia, with an estimated frequency of 1/15000 to 1/77000 births, Achondroplasia is transmitted in an autosomal dominant fashion with complete penetrance, although 80-90% of cases arise from spontaneous mutations (Andersen, 1989, Iannotti, 1994). The clinical features of heterozygous achondroplasia are very consistent among patients, and include proximal shortening of the extremities, midface hypoplasia, narrowing of the spinal column and relative macrocephaly (Rousseau, 1994, Shiang, 1994, Prinos, 1995). Final achondroplasia adult height ranges between 112 to 145 cm.
Histologically, the epiphyseal and growth plate cartilage of achondroplasia patients have a normal appearance (Rimoin, 1970). However, morphometric examinations of such patients revealed that the growth plate is shorter than normal and that the shortening is greater in homozygous than in heterozygous achondroplasia, suggesting a gene dosage effect (Horton, 1988). The intercolumnar matrix of achondroplasia patients is more abundant than normal, and focus of vascularization and transverse tunneling of the cartilage (ingrowth of blood vessels) was observed in some cases. In addition, marked periosteal bone formation was observed (Rimoin, 1970). The underlying mechanism of achondroplasia is believed to be a defect in the maturation of long bones growth plate chondrocytes (Ponseti, 1970, Maynard, 1981, Iannotti, 1994).
Achondroplasia was recently shown to be caused by point mutations in the transmembrane domain of fibroblast growth factor receptor 3 (FGFR3, Shiang, 1994). Virtually all patients show either a G to A or a G to C conversion, changing the codon for Gly 380 to Arg. This guanosine 1138 nucleotide has been described as the most mutable nucleotide to date in the human genome (Bellus, 1995). Other mutations that have been described so far lie within the transmembrane domain (Gly 375 to Cys, Superti-Furga, 1995, Ikegawa, 1995) and within the Ig3-TM linker region (Gly 346 to Glu, Prinos, 1995).
FGFR3 is a high-affinity membrane-spanning receptor for fibroblast growth factors (FGFs). The binding of FGF to the extracellular domain of FGFR3, in the presence of heparan sulfate proteoglycans, induces the dimerization of two receptor molecules, allowing transphosphorylation of tyrosines (and possibly threonine and serine residues) within the activation loop of the intracellular tyrosine kinase domains.
Activation loop phosphorylation greatly enhances the ability of FGFR3 to autophosphrylate as well as to phosphorylate substrates which transmit biological signals into the cell leading to cell proliferation, differentiation, angiogenesis, or embryogenesis (Basilico, 1992, Friesel, 1995, Jaye, 1992, Johnson, 1993).
The role of FGFR3 in the growth plate appears to be one of negative regulation of intrinsic growth rates, since mice that are homozygous for FGFR3 null alleles (e.g., by gene knock-out) show kyphosis, scoliosis, overgrowth of long bones and enlargement of the hypertrophic zone of growth plates (Deng, 1996, Colvin, 1996). This phenotype is consistent with a normal role for FGFR3 in restraining chondrocyte proliferation (upper-hypertrophic cells) and final differentiation (lower-hypertrophic cells) at the growth plates of tubular long bones and at the sutures of the skull.
Mutations in FGFR3 and in other fibroblast growth factor receptor genes can also result in other abnormalities, including skeletal and cranial malformation syndromes (Bonaventure, 1996, Muenke, 1995, Park, 1995, Webster, 1997, Lewanda, 1996). Some of the more frequent mutations of FGFRs associated with craniosynostosis and dwarfism syndromes are listed in Table 1, below.
TABLE 1
Some FGFR mutations associated wth craniosynostosis and dwarfism
syndromes
Mutation
Syndrome
FGFR 1
Pro252Arg
Pfeiffer
FGFR2
Tyr105Cys
Crouzon
Ser252Trp
Apert
Ser252Phe(CG→TT)
Apert
Ser252Leu
Normal type crouzon
934CGC→TCT(SP→FS)
Pfeiffer
Pro253Arg
Apert
Ser267Pro
Crouzon
Insertion Gly269
Crouzon
982insTGG [insG]
Crouzon
Cys278Phe
Pfeiffer; Crouzon
1037del9 [delHIQ]
Crouzon
Deletion His287-Gln289
Crouzon
Gln289Pro
Crouzon
Trp290Gly
Crouzon
Trp290Arg
Crouzon
Trp290Cys(G→C)
Pfeiffer
Trp290Cys(G→T)
Pfeiffer
Lys292Glu
Crouzon
1119-3T→G
f
Pfeiffer
1119-2A→G
f
Pfeiffer; Apert
1119-1G→C
f
Pfeiffer
Exon III acceptor splice site
Pfeiffer
Ala314Ser
Pfeiffer
Asp321Ala
Pfeiffer
Tyr328Cys
Crouzon
Asn331Ile
Crouzon
1190ins6 [insDA]
Crouzon
Gly338Arg
Crouzon
Gly338Glu
Crouzon
Tyr340His
Crouzon
Thr341Pro
Pfeiffer
Cys342Arg
Pfeiffer; Crouzon; Jackson-Weiss
Cys342Ser(G→C)
Pfeiffer; Crouzon
Cys342Ser(T→A)
Pfeiffer; Crouzon
Cys342Tyr
Pfeiffer; Crouzon
Cys342Trp
Crouzon
Cys342Phe
Crouzon
A1a344Ala(G→A)
f
Crouzon; unclassified
Ala344Pro
Pfeiffer
Ala344Gly
Crouzon; Jackson-Weiss
Ser347Cys
Crouzon
Deletion Gly345-Pro361
a
Pfeiffer; Crouzon
Ser351Cys
Unclassified
Ser354Cys
Crouzon
1245del9[delWLT]
Crouzon
Val359Phe
Pfeiffer
1263ins6
f
Pfeiffer
Ser372Cys
Beare-Stevenson cutis gyrata
Tyr375Cys
Beare-Stevenson cutis gyrata
Gly384Arg
Unclassified
FGFR 3
Arg248Cys
Thanatophoric dysplasia type I
Ser249Cys
Thanatophoric dysplasia type I
Pro250Arg
Non-syndromic craniosynostosis
Gly346Glu
Achondroplasia
Gly370Cys
Thanatophoric dysplasia type I
Ser371Cys
Thanatophoric dysplasia type I
Tyr373Cys
Thanatophoric dysplasia type I
Gly375Cys
Achondroplasia
Gly 380 to Arg
Achondroplasia
Ala391Glu
Crouzon with acantbosis; nigricans
Asn540Lys
Hypochondroplasia
Lys650Glu
Thanatophoric dysplasia type II
Lys650Met
Novel skeletal dysplasia
Stop807Gly
Thanatophoric dysplasia type I
Stop807Arg
Thanatophoric dysplasia type I
Stop807Cys
Thanatophoric dysplasia type I
Clinical similarities had already suggested that achondroplasia is part of a continuous spectrum of diseases that are all due to mutations in FGFR3 and share a common defect (McKusick, 1973). the underlying defect in these disorders is a disruption of the normal, regulated proliferation and differentiation of chondrocytes, which takes place at the epiphyseal plates of long bones and base of skull during osteogenesis. They are characterized clinically by skeletal deformities and varying degrees of dwarfism apparent before birth, typically with disproportion between the lengths of the trunk and the limbs (Horton, 1993).
On the mild side of the spectrum is hypochondroplasia, a condition associated with moderate, but variable disproportionate shortness of limbs. The trunk is normal and the face is otherwise unremarkable. The head may be normal or slightly enlarged with mild frontal bossing. The hands and feet tend to be broad and stubby. Radiographically, changes typically seen in achondroplasia are present in very mild degree. Mild shortening of the long bones with slight metaphyseal flaring are observed. The femoral necks are short and broad. The fibulae are disproportionately long, and the ilia are short and square. Shortening of the lumbar pedicles is mild to moderate and the interpediculate distance from L1 to L5 may narrow slightly.
Histologically, the growth plates of hypochondroplasia patients show no consistent microscopic abnormalities (Sillence, 1979). Some patients with hypochondroplasia have been shown to be linked to chromosome 4p, the locus where FGFR3 resides, and to have a mutation within the intracellular kinase domain of the receptor (Asn 540 to Lys, Bellus, 1995). However, other patients were shown not to be linked to chromosome 4p, and thus hypochondroplasia may be determined by mutations in genes other than FGFR3 (Stoilov, 1995).
On the lethal side of the spectrum is thanatophoric dysplasia

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