Download Lecture 1: Introduction

Natural History of
Achondroplasia
Autosomal Dominant Gene
Mutation
Achondroplasia
The most common hereditary form of
dwarfism.
Incidence rate is between 1 in 15,000 and
1 in 40,000 live births.
It is a fully penetrant autosomal dominant
disorder and the majority of cases (7580%) are the result of a new (de novo)
mutation.
Achondroplasia
 Affected individuals have short stature in arms and legs
but not torso.
 Other skeletal problems include trident hands, midfacial
hypoplasia, prominent forehead (frontal bossing),
thoracolumbar gibbus (hunched back), true
megalencephaly, and narrowing of the spaces between
the pedicles of the vertebra.
 Overall survival and the average life expectancy for ACH
population are decreased by 10 years.
 ACH individuals are at greater risk for heart problems.
 Bone abnormalities of the spine like narrow foramen
magnum and spinal canal stenosis affect mortality at all
ages but particularly in children.
 Most individuals have normal intelligence.
Achondroplasia
 Autosomal Dominant Point
mutation in FGFR3
(Fibroblast growth factor
receptor 3)
 Gene located on
Chromosome 4p16.3
 OMIM: #100800
 OMIM: *134934
 http://www.ncbi.nlm.nih.gov
/entrez/dispomim.cgi?id=10
0800
Identifying Chromosomal Location
of the ACH mutation
 Velinov et al. (Nature Genetics 6, 314 - 317
(1994) ) mapped the achondroplasia gene near
the telomere of the short arm of chromosome 4
(4p16.3), by family linkage studies using 14
pedigrees.
 A positive lod score of z=3.35 with no
recombinants was obtained with an intragenic
marker for IDUA (Apha-L-Iduronidase) mapped
earlier.
 Shiang et al. and by Rousseau et al. used the
“Candidate Gene Approach” to specifically identify
the gene involved.
 Look in the sequences in the region for a gene which
could biologically make sense as the cause of
Achondroplasia
Growth Factor
A naturally occurring protein capable of
stimulating cellular proliferation and cellular
differentiation.
Growth factors are important for regulating a
variety of cellular processes by promoting cell
differentiation and maturation.
Growth factors act as signaling molecules
between cells by binding to specific receptors
on the surface of their target cells.
Growth Factor Receptors
 Receptors are protein molecules
embedded in either the plasma
membrane or cytoplasm of a
cell, to which a mobile signaling
(or "signal") molecule may
attach.
 A molecule which binds to a
receptor is called a "ligand," and
may be a peptide (such as a
neurotransmitter), a hormone, a
pharmaceutical drug, or a toxin.
 Growth Factor Receptors have
Growth Factors as their ligands.
 When the GF binds the
receptor, a cellular response is
initiated, resulting in cellular and
developmental changes.
Transmembrane Domain of a
receptor usually has
hydrophobic amino acid
residues
What is a Fibroblast?
 A type of cell that
synthesizes and maintains
the extracellular matrix of
animal tissues.
 Provides a structural
framework for many tissues,
and plays a critical role in
wound healing.
 The most common cells of
connective tissue in
animals.
 Used in cell culture
extensively.
There are Four Fibroblast Growth
Factor Receptors
The FGF receptors bind to members of the
fibroblast growth factor family of proteins.
Growth factors are proteins which attach to cell
receptors, eliciting a response from the cell.
This response varies depending upon the cell
type, the receptor type, and the growth factor
type.
FGF Receptors have an extracellular, binding
domain with three immunoglobulin-like
domains, a single helix domain that crosses the
cell’s membrane, and an intracellular domain
with tyrosine kinase activity.
Four different genes are currently known to
encode distinct high-affinity FGF receptors.
The 4.4-kb cDNA of the FGFR3 gene
contains an open reading frame of 2520
nucleotides and consists of 19 exons and 18
introns.
There are at least 18 Fibroblast
Growth Factors (FGF)
The FGFs regulate cell proliferation,
differentiation, motility, and angiogenesis in
embryonic development.
FGF growth factors bind to their FGF receptors
in association with heparan sulphate
proteoglycan (HSPG). When this happens, the
receptors dimerize and phosphorylate
themselves.
The dimers trigger phosphorylation (P) of
downstream target proteins.
Phosphorylation
Protein kinases catalyze phosphorylation, and
phosphatases reverse the process.
Adding a phosphoryl group can change a
nonpolar hydrophobic protein into a polar, very
hydrophilic molecule – in essence, changing its
entire nature.
Each phosphorylation reaction and its reverse
requires ATP to power it.
Tyrosine phosphorylation is not as common as
other types of amino acid phosphorylation and
can be studied with specific antibodies.
What is the Achondroplasia
Mutation?
The mutation occurs in nucleotide position 1136
of the cDNA. The mutation is either a , a G-A
transition or a G-C transversion on chromosome
4.
150 of 154 unrelated achondroplasts had the GA transition and only three had a G-C
transversion at nucleotide 1138 of the FGFR3
gene.
Nucleotide 1138 of the FGFR3 gene is
considered as the most sensitive point for
germline mutation in the entire human genome.
The mutation rate is estimated to be 0.000014
per gamete per generation.
Shiang et al. 1994 Cell 78(2):335-42.
Both mutations lead to the same
Amino Acid substitution
 Reverse Transcriptase PCR product was sequenced
from heterozygous and homozygous ACH individuals
revealed a point mutation within this region in the ACH
individuals.
 Both mutations result in the substitution of an arginine
residue for a glycine at position 360 of the mature
protein, which is in the transmembrane domain of
FGFR-3.
 A mother and daughter were reported with a new
heterozygous double mutation at the same codon 380,
which substituted a lysine instead of the usual arginine.
These patients displayed a milder phenotype than the
one encountered during achondroplasia
Increased Paternal Age is
Associated with this Mutation
80% of Achondroplasia occurs with no family
history. The mutation is associated with an
increased paternal age at the time of
conception. It has been demonstrated that the
mutated allele is always from a paternal origin.
Several diseases other than skeletal dysplasias
are also associated with somatic mutations in
FGFR3.
 Seborrheic keratoses (skin growths)
 Epidermal nevi (freckles)
 Urothelial carcinomas (cancer of skin lining urethra)
RFLP Mapping can diagnose the
two different mutations
Fragment sizes: 57 bp 107 bp
AGC TAC CGG GTG
G to C transversion
Msp I
Fragment size 164 bp
AGC TAC GGG GTG
Normal Allele
Fragment sizes 55 bp 109 bp
AGC TAC AGG GTG
G to A Transition
Sfc I
RFLP of PCR Product
 PCR products of 164 BP
were amplified and
electrophoresed on a 6%
nondenaturing
polyacrylamide gel. There
are no Sfc l sites in the
normal sequence in the
FGF-DT PCR product.
 However, the G+A
transition mutation creates
an Sfc l site that, if digested,
results in fragments of 55
and 109 bp.
Role of FGFR3 in Development
Outside of the developing central nervous
system, the highest level of FGFR3
mRNAs is in the prebone cartilage
rudiments of all bones.
During endochondral ossification, FGFR3
is detected in resting but not growing
(hypertrophic) cartilage.
Endochondrial Ossification:
Conversion of Hyaline Cartilage to Bone
Calcified cartilage forms
Osteoblasts
Chondroblasts
http://en.wikipedia.org/wiki/Image:Bone_growth.png
Richette et al. Joint Bone Spine 75 (2008) 125-130
Mutant Achondroplasia Mice have
Skeletal Defects
http://www.informatics.jax.org/greenbook/figures/figure8-1H.shtml
Homozygous Achondroplasia
The presence of two alleles for achondroplasia
causes a serious skeletal disorder that leads to
early death from breathing failure due to
constriction by a tiny chest cage and
neurological problems from hydrocephalus.
Homozygous achondroplasia, although fatal,
has led to insights into other medical
conditions.
Similarities were noticed between homozygous
achondroplasia and a fatal condition of
newborns called thanatophoric dwarfism.
Achondroplasia and thanatophoric dwarfism are
due to different mutations in the FGFR 3 gene.
Other FGFR 3 Mutations
Associated with Skeletal Defects
TD = thanatophoric dysplasia, Craniosyn = craniosynostosis,
Achon = achondroplasia, Hypochon = hypochondroplasia.
Horton et al. 1998 Cells and Materials, 8: 83-87
Dwarf Horses and Dogs
Genetics of these animals is not due to the
same mutation in FGFR 3 analagous gene.
http://www.cellmigration.org/resource/komouse/images/mousefig1.png
Knockout Mice for Achondroplasia
revealed New Insights
The knock-out mouse model is missing the
FGFR 3 receptor. The negative regulation of
bone formation is lost. The result is a mouse
with excessively long bones and elongated
vertebrae, resulting in a long tail.
Mutations of FGF3R confer a "gain of function".
It is proposed that the normal function of FGFR3
is to slow down the formation of bone by
inhibiting the proliferation of chondrocytes, the
cells that produce cartilage.
The mutation increases the activity of FGFR3,
severely limiting bone growth.
Gain of Function Mutations
New or enhanced activity of a protein
Loss of function is more common
mutation
Can you think of another Human mutation
which could lead to a gain of new protein
function?
Knockout Mice with excessively long
bones, elongated vertebrae, long tails
Deng et al. 1996 Cell, Vol. 84, 911–921
CATSHL Syndrome resembles
Knockout Mice for FGFR 3
Toydemir, EM Am J Hum Genet. 2006 November; 79(5): 935–941.
Autosomal Dominant in CATSHL
Pedigree
Toydemir, EM Am J Hum Genet. 2006 November; 79(5): 935–941.
Current Medical Treatments for
Achondroplasia
Surgical limb-lengthening procedures
 Complications earlier, but more recently,
more favorable and significant increases in
height have been obtained over an 18-24month period
Human growth hormone
 Body disproportion reported, but more
recently studies show improved height
without adverse effect on trunk-leg
disproportion
Richette et al. Joint Bone Spine 75 (2008) 125-130
Future Therapies
Counteract the overactive FGFR3 effects on
endochondral bone formation.
C-type natriuretic peptide (CNP)
 Overexpression of CNP in mutant mice
chondrocytes rescues achondroplasia through a
MAPK-dependent pathway
Selective inhibition of the FGFR3 tyrosine
kinase.
 Drugs like imatinib used in cancer chemotherapy
blocking antibodies in order to interfere with
binding of FGF ligands to FGFR3
Richette et al. Joint Bone Spine 75 (2008) 125-130
Prenatal Diagnosis and Genetic
Counseling
 Some obstetric risk for mother and child
 Fetal diagnosis of achondroplasia is made with
certainty when one or both parents have condition.
 Diagnosis of achondroplasia is usually first
suspected late in gestation on the basis of longbone
foreshortening incidentally discovered by
ultrasonography.
 Disproportionately short limbs are seen in a
heterogeneous group of conditions. Misdiagnosis can lead
to inaccurate prenatal counseling.
 Genetic confirmation can be performed with chorionic villi
sampling or amniocentesis- with some risk to fetus.
Recently a noninvasive maternal blood test has been
developed.
Richette et al. Joint Bone Spine 75 (2008) 125-130