Hutchinson-Gilford Progeria

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Hutchinson-Gilford Progeria

• Miranda Lange
• Biol 409

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Introduction Background

• There are a wide range of progeroid syndromes
• Unimoidal Progeroids - only impact a single
  tissue.
• For Example
• Alzheimers it only impacts the brain tissue.
• Segmental Progeroids - much smaller subset of
  single-gene mutations that impact multiple
  aspects of the complex aging phenotype.
• For Example
• Hutchinson-Gilford syndrome (HGPS) an
  idiopathic disorder.
• The discovery of the underlying genetics of the
  HGPS model of premature aging may help to shed
  new light on humans' normal aging process (2).

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What is it?

• HGPS was named after Dr. Jonathan Hutchinson, who
  first described the disease in 1886, and Dr.
  Hastings Gilford who did the same in 1904.
• It is an exceedingly rare but typical progeria,
  characterized by
• postnatal growth retardation

• short stature ? short stature of normal aging
  is acquired in HGPS it is developmental.
• musculoskeletal abnormalities

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What is it?

• midface hypoplasia
• micrognathia
• premature atherosclerosis
• Alopecia

• Osteodysplasia with osteolysis and pathologic
  fractures
• Etc.

• Estimated to affect one in 8 million newborns
  worldwide.

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What is it?

• Many common features of aging are not seen in
  HGPS.
• For Example
• cognitive decline, age-related hearing loss and
  visual impairment, etc.
• Affected newborns usually appear normal. Within
  a year, growth rate slows dramatically, and soon
  their height and weight are significantly below
  the average.

• Median age at death is 13 years, usually due to
  coronary artery disease.

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Problem Found

• In 2003 it was discovered that HGPS is caused by
  a point mutation in a single gene, LMNA (2).
• HGPS is not hereditary. The gene change is a
  rare, chance occurrence (3).
• Children with other types of progeroid syndromes
  (not HGPS) may have diseases that are hereditary.

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Wheres the Problem?

• The LMNA gene codes for two proteins, lamin A and
  lamin C, that play a key role in stabilizing the
  inner membrane of the cell's nucleus.
• Lamin A is the structural scaffolding that holds
  the nucleus of a cell together (3).
• The responsible mutation causes LMNA to produce
  an abnormal form of lamin A.
• Abnormal lamin A destabilizes the cell's nuclear
  membrane in a way that is particularly harmful to
  tissues subjected to intense physical force, such
  as the cardiovascular and musculoskeletal systems
  (Fig. 1) (2).

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Figure 1.
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
Immunofluorescence results showing the
abnormal cellular shape in HGPS children (e-h)
compared to normal cells (a-d). Antibodies used
directed against lamin A.
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Experimentation Synopsis
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297

• HGPS was shown to be caused by de novo dominant
  mutations in LMNA, resulting in the in-frame
  deletion of 50 amino acids near the carboxyl
  terminus of the encoded lamin A (6).
• What was done?
• A whole-genome scan including 403 polymorphic
  microsatellite markers with an average spacing of
  9.2 cM was performed on 12 DNA samples derived
  from individuals representing classical HGPS.

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Experimentation Synopsis
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297

• What was found?
• Two HGPS samples with genotypes 46,XY UPD
  (1Xp11.2qter) and 46,XX UPD (1)(q22qter),
  respectively, were found to have uniparental
  isodisomy (UPD) of chromosome 1.
• Spectral karyotyping and G-banding of one UPD
  case showed a normal karyotype.
• NOTE mouse model found in Mounkes et al.(2003).
  Nature, Vol. 423, p. 298-

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Fibroblast Cultures
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297

• A fibroblast culture was obtained from one
  individual (sample I.D. C8803) and his parents
  the first steps of positional cloning.
• Genotyping of microsatellite markers identified a
  6 Mb interval where all tested paternal alleles
  were completely missing a deletion (Fig. 2,a).
• Using fluorescent in situ hybridization (FISH)
  with bacterial artificial chromosomes (BACs) that
  map throughout the 6 Mb interval, the deletion
  was also found present in cells with apparently
  normal karyotype. (Fig. 2,b).

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Figure 2,a.
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
The boxed interval is the region in proband
C8803 that has been inherited exclusively from
the mother. NA not available.
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Figure 2,b.
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
FISH analysis of a metaphase spread from C8803
fibroblasts, hybridized with BAC probes
RP11-110J1 (green) and RP11-91G5 (red).
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Fibroblast Cultures
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297

• Putting all of the information together with
  genotypes from 44 additional microsatellite
  markers, it was determined that the HGPS gene
  must lie in an interval of 4.82 Mb on chromosome
  1q (Fig. 2,c).
• The candidate interval contains roughly 80 known
  genes.
• Time for the second step of positional cloning
  identifying candidate genes and, thus, coding
  regions (exons).

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Figure 2,c.
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
Summary map of the candidate region.
Microsatellite markers are indicated with arrows
horizontal bars indicate BAC probes that were
used for FISH on sample C8803. LMNA is one of
the approximately 80 known genes in the 4.82-Mb
candidate interval.
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Exons Introns
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297

• LMNA contains 12 exons and covers about 25 kb of
  genomic DNA.
• Lamin A is coded by exons 112 and lamin C by
  exons 110.
• A splice site within exon 10, located just
  upstream of the stop codon for lamin C, splices
  together with exons 11 and 12 to code for lamin A.

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PCR Amplification
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297

• Step three in positional cloning identify
  specific gene(s) responsible for the mutant
  phenotype, assaying for RNA expression.
• PCR amplification of all of the exons of the
  mutant LMNA gene (including exonintron
  boundaries), followed by direct sequencing, was
  carried out in 23 samples.
• In 18 of these samples, a heterozygous base
  substitution within exon 11 of the LMNA was
  identified (Fig. 4).

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Figure 4
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
Point mutations in exon 11 of LMNA cause HGPS.
Sequence traces from a normal control and two
HGPS patients with heterzygous base
substitutions, one within the same codon
(G608S(GGC . AGC)), and the other within exon 2
(E145K(GAG . AAG)).
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Table 1.
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
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Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
How do these de novo mutations cause HGPS?

• Cloning and sequencing of the normal full-length
  RNA containing the codon mutation showed that 7
  out of 23 clones carry the mutant sequence.
• Therefore, activation of the cryptic splice site
  within exon 11 is not complete in mutants.
• To test RT-PCR using a forward and reverse
  primer and Northern blot (Fig. 5,a.).
• RNA samples from cell lines with HGPS mutations -
  additional, smaller product appears.
• Sequencing fragments shows that 150 nucleotides
  within exon 11 are missing the elusive
  deletion.

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Figure 5,a.
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
MW Markers
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Is the Mutant Expressed?
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297

• To test Western blot using a monoclonal
  antibody against lamin A/C (Fig. 5,b).
• In addition to the normal lamin A/C bands, an
  additional band is found in four lanes containing
  samples from classical HGPS cases.
• Yes, the mutant is expressed, further proving
  that HGPS is the result of the mutation.

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Figure 5,b.
Eriksson, et al. (2003). Nature, Vol. 423, p.
293-297
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Is there a test for Progeria?

• YES!! A DNA diagnostic test!
• Peviously HGPS could only be diagnosed from an
  overall look and X-rays (misdiagnosis was
  frequent).
• The new genetic test can definitively identify
  HGPS and produce earlier diagnosis, fewer
  misdiagnoses, and early medical intervention (3).
  
• Samples taken from the blood and skin cells of
  HPGS children are sequenced to provide the
  definitive diagnosis, assuring scientists they
  are truly working with progeria cells.

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How can it be treated?

• Nutrition (9)
• Nutrition is a very difficult daily aspect
  because HGPS children often have extremely poor
  appetites. Thus, improved caloric intake may
  result in better health, improved energy level
  and mood, and improved quality of life.
• Low Doses of Aspirin (10)
• Children with HGPS are at high risk for heart
  attacks and thrombotic strokes.
• Low doses of aspirin can help prevent
  atherothrombotic events by inhibiting platelet
  aggregation.
• Physical and Occupational Therapy (11)

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Resources

• George M. Martin, Junko Oshima (2000). Lessons
  from Human Progeriod Syndromes. Nature, Vol. 408,
  p. 263-266
• National Human Genome Research Institute (2004).
  Learning About Progeria What we Know about
  Heredity and Progeria. http//www.genome.gov/1100
  7255
• The Progeria Research Foundation, Inc (2003).
  What is Progeria? The Science Behind Progeria.
  http//www.progeria research.org/
• Maria Eriksson, W. Ted Brown, Leslie B. Gordon,
  Michael W. Glynn, Joel Singerk, Laura Scottk,
  Michael R. Erdos, Christiane M. Robbins, Tracy Y.
  Moses, Peter Berglund, Amalia Dutra, Evgenia Pak,
  Sandra Durkin, Antonei B. Csoka, Michael
  Boehnkek, Thomas W. Glover, Francis S. Collins
  (2003). Recurrent de novo Point Mutations in
  Lamin A cause HutchinsonGilford Progeria
  Syndrome. Nature, Vol. 423, p. 293-297
• Annachiara De Sandre-Giovannoli, Rafaelle
  Bernard, Pierre Cau, Claire Navarro, Jeanne
  Amiel, Irene Boccaccio, Stanislas Lyonnet, Colin
  L. Stewart, Arnold Munnich, Martine Le Merrer,
  Nicolas Levy (2003). Lamin A Trucation in
  Hutchinson-Gilford Progeria. Science, Vol. 300,
  p. 2055
• Csoka AB, English SB, Simkevich CP, Ginzinger DG,
  Butte AJ, Schatten GP, Rothman FG, Sedivy JM
  (2004). Genome-scale Expression Profiling of
  Hutchinson-Gilford progeria syndrome Reveals
  Widespread Transcriptional Misregulation Leading
  to Mesodermal/Mesenchymal Defects and Accelerated
  Atherosclerosis. Aging Cell., Vol. 3(4), p.
  235-43
• Pulkkinen, L., Bullrich, F., Czarnecki, P.,
  Weiss, L. Uitto, J. (1997). Maternal
  Uniparental Disomy of
• Chromosome 1 with Reduction to Homozygosity of
  the LAMB3 Locus in a Patient with Herlitz
  Junctional Epidermolysis Bullosa. Am. J. Hum.
  Genet., Vol. 61, p. 611619
• Gelb, B. D. et al (1998). Paternal Uniparental
  Disomy for Chromosome 1 Revealed by Molecular
  Analysis of a Patient with Pycnodysostosis. Am.
  J. Hum. Genet., Vol. 62, p. 848854

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Resources

• Harten, Ingrid A., Hardy, Christine, Gordon,
  Leslie B. (2002). Nutritional Supplements In
  Hutchinson-Gilford Progeria Syndrome
  Information for Families and Caregivers From The
  Progeria Research Foundation. The Progeria
  Research Foundation, Inc.
• Gordon, Leslie B., Feit, Lloyd R., Smoot, Leslie
  B. (2002). Important Information For You and
  Your Doctors About Low-Dose Aspirin Treatment and
  Progeria Information for Families and Caregivers
  From The Progeria Research Foundation. The
  Progeria Research Foundation, Inc.
• Gordon, Leslie B., MacDonnell, Lisa (2004).
  Physical Therapy and Occupational Therapy in
  Progeria Information for Families and
  Caretakers From The Progeria Research Foundation.
  The Progeria Research Foundation, Inc.
• Mounkes, Leslie C., Kozlov, Serguei, Hernandez,
  Lidia, Sullivan, Teresa, and Stewart, Colin L.
  (2003). A Progeroid Syndrome in Mice is Caused
  by Defects in A-type Lamins. Nature, Vol. 423, p.
  298-