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Francis S. Collins, M.D., Ph.D. July 28, 2003
Mutations in Lamin A are the Cause of Hutchinson-Gilford Progeria Syndrome Francis S. Collins, M.D., Ph.D. July 28, 2003
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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)
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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
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Case #1 46,XY UPD (1)(p11.2;qter) Case #2 46,XX UPD (1)(q22;qter)
NA NA NA 1p D1S252 1p D1S2696 1q D1S2344 1q D1S2222 1q D1S498 1q D1S2347 1q D1S2346 1q D1S1153 1q D1S1653 1q D1S2635 1q D1S196 1q D1S2791 1q D1S2127 1q D1S191 1q D1S413 1q D1S2685 1q D1S2141 1q D1S2763 1q D1S2800 1q D1S2850 1q D1S2836 304 92 106 99 107 1p11.2 1q22
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Earlier report of an inverted insertion on chromosome 1q in sample C8803: A monozygotic twin with severe HGPS 1q23 1q32 1q44 1q32 1q23 30 % 70% 46 XY, inv ins (1;1)(q32;q44q23) Brown et al AJHG, 1990
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Paternal deletion in sample C8803
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 92 93 99 96 100 99 99 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 93 99
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Paternal deletion in C8803 < Deletion 1q21.3-q23.1 1q23 1q32 1q44
97 % 3 %
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Paternal deletion in C8803 1p 1q UPD case #1 UPD case #2 Dtetra46 Pdi3
1q21.3 D1S2346 1q23.2 D1S2635 RP1-140J1 RP11-66D17 RP11-120D12 RP11-137P24 RP11-91G5 RP11-110J1 Deletion 5.66 Mb Maximum deletion 5.9 Mb UPD case #1 UPD case #2
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Paternal deletion in C8803 1p 1q LMNA UPD case #1 UPD case #2 Dtetra46
Pdi3 1q21.3 D1S2346 1q23.2 D1S2635 RP1-140J1 RP11-66D17 RP11-120D12 RP11-137P24 RP11-91G5 RP11-110J1 Deletion 5.66 Mb Maximum deletion 5.9 Mb UPD case #1 UPD case #2
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Mutations in Lamin A/C Recessive mutations 1 2 3 4 5 6 7 8 9 10 11 12
Stop Lamin C Stop Lamin A ATG NLS 1 2 3 4 5 6 7 8 9 10 11 12 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
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R.D. Goldman et al., Genes and Development 16: 533-547, 2002
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Sequencing exon 11 of LMNA
Normal GTG GGC GGA Progeria GTG GG GGA Mother GTG GGC GGA Father GTG GGC GGA C T
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Mutations found in LMNA – Classic HGPS
Classical HGPS Codon 608 seq 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
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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
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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
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How can the observed mutations cause progeria?
Splice donor sequence A G G T A G T G Normal sequence G G T G G G C Mutation 1 G G T G G G T Mutation 2 G G T G A G C Normal 3’UTR Exon 11 12 Mutant
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RT-PCR experiment Exon 10 3’UTR Exon 11 12 Normal Exon 10 3’UTR Ex 12 Mutant 1 & 2 639 489 Mutant 1 Mutant 2
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Western and immunofluorescence with lamin A/C antibody
mito- chondria Lamin A del 50 prelamin A unaffected father Lamin C Mutant 1 Mutant 2 classical HGPS
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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)
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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?
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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
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Mouse models of human disease
Transgenics Simple Inducible BAC-based Knock-out Knock-in Conventional vs. conditional
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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
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BAC transgenic simple G608G
Start clone: RP11-702H12 Human genomic DNA insert size: kb 164.4 kb 15.2 44.3 13.5 LMNA SSR2 P (c1orf5) P RAB25 P (FLJ12287) 9.5 (11.9) 17.5 3.7 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
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BAC transgenic: G608G C-06 and negative littermate
day 24 day 30 day 30
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BAC transgenic conditional
loxP Start clone: BAC simple G608G Strain: C57Bl/6 FRT loxP 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
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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
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Maria Eriksson NIH / NHGRI University of Michigan /
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
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Injection efficiency 61 25 59 29 39 8 Construct # Pups
PCR screen- transgene positive (Fo) tet 77 (wt) 61 25 tet75 (G608G) 59 29 BAC simple G608G 39 8
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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
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Normal GTG GGC GGA Progeria GTG GG GGA 18/20 GTG GC GGA 1/20
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 /20 GTG GC GGA /20 C T G A
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