Hutchinson-Gilford Progeria

1. Hutchinson-Gilford Progeria Miranda Lange Biol 409

2. Introduction & Background • There are a wide range of progeroid syndromes: • “Unimoidal Progeroid’s” - only impact a single tissue. • For Example: • Alzheimers: it only impacts the brain tissue. • “Segmental Progeroid’s” - 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).

3. 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

4. 1 2 Fig. A 1. Radiograph of the chest demonstrates hypoplasia of the clavicles. 2. Radiograph of the pelvis demonstrates bilateral hip dysplasia and coxa valga (hip abnormalities). 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.

5. 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.

6. 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.

7. Where’s 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).

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

9. 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.

10. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Experimentation – Synopsis • What was found? • Two HGPS samples with genotypes 46,XY UPD (1Xp11.2;qter) and 46,XX UPD (1)(q22;qter), 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-

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

12. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Figure 2,a. The boxed interval is the region in proband C8803 that has been inherited exclusively from the mother. NA = not available.

13. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Figure 2,b. FISH analysis of a metaphase spread from C8803 fibroblasts, hybridized with BAC probes RP11-110J1 (green) and RP11-91G5 (red).

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

15. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Figure 2,c. 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.

16. Fig. 3. Representation of LMNA and Lamin A as blue and red colors, respectively. The deleted LMNAtranscript junction sequence is shown as a 150 bp deletion extending from G1819 to the exon 11 end (black box) and is indicated by a black bar in the Lamin A tail. The CaaX-box motif (cysteine-aliphatic-aliphatic-any amino acid) at the C terminus is subject to farnesylation (2). Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Exons & Introns • LMNA contains 12 exons and covers about 25 kb of genomic DNA. • Lamin A is coded by exons 1–12 and lamin C by exons 1–10. • 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.

17. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 PCR Amplification • 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 exon–intron 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).

18. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Figure 4 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)).

19. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Table 1.

20. 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.

21. Demonstration of the abnormal splice product using RT–PCR, showing an abnormal product of 489 bp in two HGPS probands due to activation of the cryptic splice site. Alternative lanes to the right lack reverse transcriptase. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Figure 5,a. MW Markers

22. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Is the Mutant Expressed? • 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.

23. Western blot using a monoclonal antibody against lamin A/C. Lanes 1, 5, 8 and 9 are from AG03506, AG03344, AG11498 and AG01972; all carry G608G(GGC . GGT). Lane 4 is from AG10801, carrying G608S(GGC . AGC); lanes 2 and 3 are from parents of AG03506; lanes 6 and 7 are from father of AG03259 and mother of AG06917, respectively. Lanes 1–5 are from lymphoblastoid cell lines; lanes 6–9 are from fibroblasts. A protein sample from HeLa cells is in lane 10. Eriksson, et al. (2003). Nature, Vol. 423, p. 293-297 Figure 5,b.

24. 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.

25. 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)

27. 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/11007255 • 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 Hutchinson–Gilford 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. 611–619 • 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. 848–854

28. 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-