Natural History of Achondroplasia

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Natural History of Achondroplasia

• Autosomal Dominant Gene Mutation

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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 (75-80) are
  the result of a new (de novo) mutation.

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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.

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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
  100800

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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 z3.35 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

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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.

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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
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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.

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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 cells
  membrane, and an intracellular domain with
  tyrosine kinase activity.

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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.
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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.

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(No Transcript)
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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.

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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 G-A
  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.

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Shiang et al. 1994 Cell 78(2)335-42.
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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

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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)

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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
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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 GA transition mutation creates an
  Sfc l site that, if digested, results in
  fragments of 55 and 109 bp.

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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.

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Endochondrial Ossification Conversion of Hyaline
Cartilage to Bone
Calcified cartilage forms
Osteoblasts
Chondroblasts
http//en.wikipedia.org/wiki/ImageBone_growth.png
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Richette et al. Joint Bone Spine 75 (2008) 125-130
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Mutant Achondroplasia Mice have Skeletal Defects
http//www.informatics.jax.org/greenbook/figures/f
igure8-1H.shtml
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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.

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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
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Dwarf Horses and Dogs

• Genetics of these animals is not due to the same
  mutation in FGFR 3 analagous gene.

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http//www.cellmigration.org/resource/komouse/imag
es/mousefig1.png
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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.

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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?

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Knockout Mice with excessively long bones,
elongated vertebrae, long tails
Deng et al. 1996 Cell, Vol. 84, 911921
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CATSHL Syndrome resembles Knockout Mice for FGFR 3
Toydemir, EM Am J Hum Genet. 2006 November
79(5) 935941.
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Autosomal Dominant in CATSHL Pedigree
Toydemir, EM Am J Hum Genet. 2006 November
79(5) 935941.
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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 18-24-month 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
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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
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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