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Mutations in Lamin A are the Cause of Hutchinson-Gilford Progeria Syndrome

Mutations in Lamin A are the Cause of Hutchinson-Gilford Progeria Syndrome. Francis S. Collins, M.D., Ph.D. July 28, 2003. Inheritance pattern of HGPS?. Autosomal dominant reproductive lethal Recurrence rate much less than ¼ Paternal age effect Autosomal recessive

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Mutations in Lamin A are the Cause of Hutchinson-Gilford Progeria Syndrome

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  1. Mutations in Lamin A are the Cause of Hutchinson-Gilford Progeria Syndrome Francis S. Collins, M.D., Ph.D. July 28, 2003

  2. Inheritance pattern of HGPS? • Autosomal dominant reproductive lethal • Recurrence rate much less than ¼ • Paternal age effect • Autosomal recessive • Rare cases of recurrence in sibs (but ? Dx)

  3. Initial approach Homozygosity mapping: • 407 microsatellite markers • Average spacing of 10 cM • Samples obtained from Coriell Cell Repositories and Progeria Research Foundation Result: No evidence of a region of homozygosity BUT: Surprises were uncovered

  4. Case #1 46,XY UPD (1)(p11.2;qter) Case #2 46,XX UPD (1)(q22;qter) NA NA NA 96 102 173 173 255 255 148 148 206 206 272 272 111 111 304 108 108 149 149 332 332 172 172 124 124 159 159 260 260 109 109 259 259 169 169 219 219 153 153 246 246 1p13.1 D1S252 1p11.2 D1S2696 1q21.1 D1S2344 1q21.2 D1S2222 1q21.3 D1S498 1q21.3 D1S2347 1q21.3 D1S2346 1q22 D1S1153 1q23.1 D1S1653 1q23.2 D1S2635 1q24.2 D1S196 1q25.2 D1S2791 1q25.3 D1S2127 1q31.1 D1S191 1q31.3 D1S413 1q32.2 D1S2685 1q41 D1S2141 1q42.12 D1S2763 1q42.2 D1S2800 1q43 D1S2850 1q44 D1S2836 92 106 167 169 253 255 148 148 198 202 272 284 99 107 305 305 104 104 146 146 330 330 172 172 128 128 163 163 260 260 115 115 263 263 169 169 215 215 155 155 249 249 1p11.2 1q22

  5. Earlier report of an inverted insertion on chromosome 1q in sample C8803: A monozygotic twin with severe HGPS 1q23 1q44 1q32 1q23 1q32 30% 70% 46 XY, inv ins (1;1)(q32;q44q23) Brown et al AJHG, 1990

  6. Paternal deletion in sample C8803 96 100 232 230 145 147 99 99 216 220 309 313 135 143 100 112 232 244 149 146 106 92 238 230 145 145 93 99 218 216 297 287 139 139 108 108 240 228 153 144 1p13.1 D1S252 1q21.1 D1S442 1q21.3 D1S2345 1q21.3 D1S2346 Pdi3 1q22 D1S1153 1q23.1 D1S506 1q23.1 D1S1653 Dtetra46 1q23.2 D1S2635 C8803 & C8803b P4 1p13.1 D1S252 1q21.1 D1S442 1q21.3 D1S2345 1q21.3 D1S2346 Pdi3 1q22 D1S1153 1q23.1 D1S506 1q23.1 D1S1653 Dtetra46 1q23.2 D1S2635 106 100 238 230 145 147 93 99 218 218 297 297 139 139 108 108 240 240 153 146

  7. Paternal deletion in C8803 < Deletion 1q21.3-q23.1 1q23 1q44 1q32 1q23 1q32 97 % 3 %

  8. Paternal deletion in C8803 1p 1q Dtetra46 Pdi3 1q23.2 D1S2635 1q21.3 D1S2346 RP1-140J1 RP11-66D17 RP11-120D12 RP11-91G5 RP11-137P24 RP11-110J1 Deletion 5.66 Mb Maximum deletion 5.9 Mb UPD case #1 UPD case #2

  9. LMNA Paternal deletion in C8803 1p 1q Dtetra46 Pdi3 1q23.2 D1S2635 1q21.3 D1S2346 RP1-140J1 RP11-66D17 RP11-120D12 RP11-91G5 RP11-137P24 RP11-110J1 Deletion 5.66 Mb Maximum deletion 5.9 Mb UPD case #1 UPD case #2

  10. Mutations in Lamin A/C Recessive mutations Stop Lamin A Stop Lamin C ATG NLS 1 2 3 4 5 6 7 8 9 11 12 10 Dominant mutations Familial Partial Lipodystrophy Charcot-Marie-Tooth Disease Type 2 Limb-Girdle Muscular Dystrophy Type 1B Dilated Cardiomyopathy Emery-Dreifuss Muscular Dystrophy Mandibuloacral Dysplasia

  11. R.D. Goldman et al., Genes and Development 16: 533-547, 2002

  12. Sequencing exon 11 of LMNA Normal GTG GGC GGA Progeria GTG GG GGA Mother GTG GGC GGA Father GTG GGC GGA C T

  13. Mutations found in LMNA – Classic HGPS Classical HGPS Codon608seq Mutation Comment Mother Father Sibling(s) AG01972 GGC/T G608G NA NA NA AG06297 GGC/T G608G NA NA NA AG10801 A/G GC G608S NA NA NA AG11498 GGC/T G608G NA NA NA AG11513 GGC/T G608G NA NA NA AG03506 GGC/T G608G Normal Normal Normal AG03344 GGC/T G608G Normal Normal Normal AG03259 GGC/T G608G Normal Normal Normal AG06917 GGC/T G608G Normal Normal NA AG10578 GGC UPD Normal NA Normal AG10579 GGC/T G608G NA NA NA AG10587 GGC/T G608G Normal NA ND HGADFN001 GGC/T G608G NA NA NA HGADFN003 GGC/T G608G NA NA NA AG10677 GGC NA NA NA HGALBV009 GGC/T G608G Normal Normal NA HGALBV011 GGC/T G608G Normal Normal NA HGALBV057 GGC/T G608G Normal Normal NA HGADFN005 GGC UPD NA NA NA HGADFN008 GGC/T G608G NA NA NA HGADFN014 GGC/T G608G NA NA NA HGALBV071 GGC/T G608G NA NA NA AG10548/C8803 GGC Deletion Normal Normal NA

  14. Other mutations found in LMNA in atypical HGPS • AG10677 – E145K (exon 2) • Limited loss of hair and subcutaneous tissue, severe strokes • AG07091 – R471C/R527C • Survived to age 28, phenotype partially overlaps with MAD

  15. Parental origin of mutation? • Use nearby polymorphisms and PCR to track which parental chromosome bears the G608G new mutation • In 5/5 informative cases, the mutation was paternal

  16. How can the observed mutations cause progeria? Splice donor sequence A A G G G T T G Normal sequence G G T G G G C G G T G G G T Mutation 1 G G T G A G C Mutation 2 Normal 3’UTR 12 Exon 11 Mutant

  17. RT-PCR experiment Exon 10 3’UTR Exon 11 12 Normal Exon 10 3’UTR Mutant 1 & 2 Ex 12 639 489 Mutant 1 Mutant 2

  18. Western and immunofluorescence with lamin A/C antibody lamin A/C mito- chondria Lamin A del 50 prelamin A unaffected father Lamin C Mutant 1 Mutant 2 classical HGPS

  19. Molecular basis of progeria • De novo point mutations in LMNA are the cause of Hutchinson-Gilford progeria syndrome • The G608G mutation accounts for ~90% of cases • The mutation induces an abnormal splice event that deletes 150 nt from the coding region of the RNA • The mutant protein, progerin, lacks 50 aa near the C-terminus • Two cases of segmental UPD from fibroblast DNA do not show the mutation -- we postulate that this is a somatic rescue event (in vitro or in vivo)

  20. Some big questions about HGPS • How does progerin affect the structure of the nuclear lamina so dramatically? • How does this lead to the phenotype? • How can this information be used to develop new ideas about therapy? • Could the LMNA gene play a role in normal aging?

  21. Testing the role of LMNA in normal aging • Identify single nucleotide polymorphisms across the gene • Look at allele and haplotype frequencies for all of these in • 250 centenarians • 250 matched controls

  22. Mouse models of human disease • Transgenics • Simple • Inducible • BAC-based • Knock-out • Knock-in • Conventional vs. conditional

  23. Lamin A minigene-tet inducible transgene cDNA lamin A exon 1- exon11+ intron 11 and exon 12 Strain: Fvb Hind III Hind III Ase I Not I ATG TAA * tet75 Tet-op IRES eGFP SV40/pA G608G Hind III Hind III Ase I Not I ATG TAA tet77 Tet-op IRES eGFP SV40/pA Wt ATG TAA * Tet-op IRES eGFP SV40/pA G608G Activation of transgene X ie. Tissue specific transactivator and tetracycline

  24. BAC transgenic simple G608G Start clone: RP11-702H12 Human genomic DNA insert size: 164.4 kb 164.4 kb 15.2 44.3 13.5 LMNA SSR2 P (c1orf5) P RAB25 P (FLJ12287) 3.7 9.5 (11.9) 17.5 9.3 25.4 22.2 (24.1) BAC simple G608G Strain: C57Bl/6 Xba I Xba I LMNA ATG FRT TAA * P P RAB25 G608G 25.5

  25. day 30 day 24 day 30 BAC transgenic: G608G C-06 and negative littermate

  26. BAC transgenic conditional loxP loxP FRT Start clone: BAC simple G608G Strain: C57Bl/6 TAA Shuttle fragment X X kan 212 Wt 367 LMNA ATG TAA * P RAB25 G608G Recombineering and flpe LMNA loxP loxP ATG TAA TAA * Wt G608G X Tissue specific-cre expressing mice LMNA ATG TAA * P RAB25 G608G

  27. Conclusions • HGPS is due to gain of function mutations in LMNA • Identification of the molecular basis now allows accurate diagnosis • Cellular and organismal pathophysiology can now be explored in detail • We are fortunate to have landed on a gene where so much good science has already been done! • A major area of focus should now be on developing possible means of treatment

  28. NIH / NHGRI Maria Eriksson Michael Erdos Jun Cheng Lisa Garrett Christiane Robbins Peter Chines Amalia Dutra Evgenia Pak Elizabeth Gillanders University of Michigan / Department of Biostatistics Michael Boehnke Joel Singer Laura Scott Elixir Pharmaceuticals Alan Watson University of Michigan / Department of Human Genetics Thomas Glover Michael Glynn Sandra Durkin Tony Csoka The Progeria Research Foundation Leslie Gordon NY State Institute for Basic Research in Developmental Disabilities Ted Brown Coriell Cell Repositories Children's Hospital Oakland Pieter de Jong Yuko Yoshinaga Kazutoyo Osoegawa

  29. Injection efficiency

  30. BAC simple G608G founders • Weight at day 30: • A-04 (F): 21.7 g • B-02 (M): 24.8 g • B-06 (F): 20.5 g • C-02 (M): 22.2 g • C-05 (F): 20.0 g • C-06 (F): 15.6 g • D-01(F): 18.1 g • F-06 (F): 19.4 g • Neg litter mates: (M) 22.1 g and (F)19.0 g

  31. Nearly all cases of progeria have a de novo mutation in codon 608 of the lamin A/C gene Normal GTG GGC GGA Progeria GTG GG GGA 18/20 GTG GC GGA 1/20 607 608 609 C T G A

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