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– Rare cases of recurrence in sibs (but ? Dx)

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

Case #1 46,XY UPD (1)(p11.2;qter)

1p13.1 D1S2521p11.2 D1S26961q21.1 D1S23441q21.2 D1S22221q21.3 D1S4981q21.3 D1S23471q21.3 D1S23461q22 D1S11531q23.1 D1S16531q23.2 D1S26351q24.2 D1S1961q25.2 D1S27911q25.3 D1S21271q31.1 D1S1911q31.3 D1S4131q32.2 D1S26851q41 D1S21411q42.12 D1S27631q42.2 D1S28001q43 D1S28501q44 D1S2836

96 102173 173255 255148 148206 206272 272111 111304 304108 108149 149332 332172 172124 124159 159260 260109 109259 259169 169219 219153 153246 246

Case #246,XX UPD (1)(q22;qter)

92 106167 169253 255148 148198 202272 284 99 107305 305104 104146 146330 330172 172128 128163 163260 260115 115263 263169 169215 215155 155249 249

1q22

1p11.2

NA NANA

Earlier report of an inverted insertion on chromosome 1q in sample C8803: A monozygotic twin with severe HGPS

46 XY, inv ins (1;1)(q32;q44q23)

Brown et al AJHG, 1990

70%

30 %

1q32

1q32

1q44

1q23

1q23

1p13.1 D1S2521q21.1 D1S4421q21.3 D1S23451q21.3 D1S2346 Pdi31q22 D1S11531q23.1 D1S5061q23.1 D1S1653 Dtetra461q23.2 D1S2635

1p13.1 D1S2521q21.1 D1S4421q21.3 D1S23451q21.3 D1S2346 Pdi31q22 D1S11531q23.1 D1S5061q23.1 D1S1653 Dtetra461q23.2 D1S2635

C8803 & C8803b P4

106 92238 230145 145 93 99218 216297 287139 139108 108240 228153 144

96 100232 230145 147 99 99216 220309 313135 143100 112232 244149 146

106 100238 230145 147 93 99218 218297 297139 139108 108240 240153 146

Paternal deletion in sample C8803

Paternal deletion in C8803

3 %

1q32

1q32

1q44

1q23

1q23

97 %

< Deletion 1q21.3-q23.1

Pdi3

Deletion 5.66 Mb

Maximum deletion 5.9 Mb

Dtetra461q21.3 D1S2346

1q23.2 D1S2635

1p 1q

RP11-120D12

RP11-91G5

RP11-66D17

RP11-110J1

RP1-140J1

RP11-137P24

Paternal deletion in C8803

UPD case #1

UPD case #2

Pdi3

Deletion 5.66 Mb

Maximum deletion 5.9 Mb

Dtetra461q21.3 D1S2346

1q23.2 D1S2635

1p 1q

RP11-120D12

RP11-91G5

RP11-66D17

RP11-110J1

RP1-140J1

RP11-137P24

Paternal deletion in C8803

UPD case #1

UPD case #2

LMNA

1 32 4 5 6 7 8 9 10 11 12

Mutations in Lamin A/C

Stop Lamin C

NLS

Stop Lamin AATG

Emery-Dreifuss Muscular Dystrophy

Limb-Girdle Muscular Dystrophy Type 1B

Familial Partial Lipodystrophy

Recessive mutations

Dominant mutations

Dilated Cardiomyopathy

Charcot-Marie-Tooth Disease Type 2

Mandibuloacral Dysplasia

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

Sequencing exon 11 of LMNA

Normal GTG GGC GGA

Progeria GTG GG GGA

Mother GTG GGC GGA

Father GTG GGC GGA

CT

Classical HGPS Mutation Comment Mother Father Sibling(s)AG01972 GGC/T G608G NA NA NAAG06297 GGC/T G608G NA NA NAAG10801 A/G GC G608S NA NA NAAG11498 GGC/T G608G NA NA NAAG11513 GGC/T G608G NA NA NAAG03506 GGC/T G608G Normal Normal NormalAG03344 GGC/T G608G Normal Normal NormalAG03259 GGC/T G608G Normal Normal NormalAG06917 GGC/T G608G Normal Normal NAAG10578 GGC UPD Normal NA NormalAG10579 GGC/T G608G NA NA NAAG10587 GGC/T G608G Normal NA NDHGADFN001 GGC/T G608G NA NA NAHGADFN003 GGC/T G608G NA NA NAAG10677 GGC NA NA NAHGALBV009 GGC/T G608G Normal Normal NAHGALBV011 GGC/T G608G Normal Normal NAHGALBV057 GGC/T G608G Normal Normal NAHGADFN005 GGC UPD NA NA NAHGADFN008 GGC/T G608G NA NA NAHGADFN014 GGC/T G608G NA NA NAHGALBV071 GGC/T G608G NA NA NAAG10548/C8803 GGC Deletion Normal Normal NA

Mutations found in LMNA – Classic HGPS

Codon 608 seq

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

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

Splice donor sequence

TGG AG

A G T

Normal sequence

Mutation 1

Mutation 2

G G T G G G C

G G T G G G T

G G T G A G C

How can the observed mutations cause progeria?

3’UTR12

Normal

Exon 11

Mutant

3’UTR12Exon 11Exon 10

Mutant 1 & 23’UTR12

Normal

Exon 10

RT-PCR experiment

Ex

Mutant 1

489 639

Mutant 2

Western and immunofluorescence with lamin A/C antibody

Lamin A

Lamin Cdel 50 prelamin A

Mutant 1 Mutant 2

mito-chondria

lamin A/C

unaffected father

classical HGPS

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)

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?

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

Mouse models of human disease

• Transgenics– Simple– Inducible– BAC-based

• Knock-out• Knock-in• Conventional vs. conditional

Lamin A minigene-tet inducible transgene

ATGATG TAATAA

**G608GG608G

Tet-opTet-op IRESIRES eGFPeGFP SV40/pASV40/pA

Ase IAse I Not INot I

ATGATG TAATAA

WtWt

Tet-opTet-op IRESIRES eGFPeGFP SV40/pASV40/pA

Ase IAse I Not INot I

tet75

tet77

Hind IIIHind III Hind IIIHind III

Hind IIIHind III Hind IIIHind III

cDNA lamin A exon 1- exon11+ intron 11 and exon 12Strain: Fvb

ATGATG TAATAA

**Tet-opTet-op IRESIRES eGFPeGFP SV40/pASV40/pA

X

G608GG608G

ie. Tissue specific transactivator and tetracycline

Activation of transgene

BAC transgenic simple G608G

ATGATG TAATAA

LMNA

Start clone: RP11-702H12 Human genomic DNA insert size: 164.4 kb

FRT

G608G*

Xba IXba I Xba IXba I

P RAB25P (c1orf5)SSR2 P (FLJ12287)

164.4 kb

25.4

13.5

22.2 (24.1)

LMNA44.3

9.33.717.59.5 (11.9)

15.2

BAC simple G608GStrain: C57Bl/6

25.5

P P RAB25

BAC transgenic: G608G C-06 and negative littermate

day 30

day 30

day 24

BAC transgenic conditional

ATGATG TAATAA

LMNA

G608G*

Wtkan

TAATAA

X XloxP loxP

ATGATG TAATAA

LMNA

G608G*

Recombineering and flpe

TAATAA

Wt

loxP loxP

X Tissue specific-cre expressing mice

ATGATG TAATAA

G608G*

Start clone: BAC simple G608G Strain: C57Bl/6

LMNA

P

P RAB25

RAB25

212 367

FRTShuttlefragment

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

NIH / NHGRI

Maria ErikssonMichael ErdosJun ChengLisa GarrettChristiane RobbinsPeter ChinesAmalia DutraEvgenia PakElizabeth Gillanders

University of Michigan / Department of Biostatistics Michael BoehnkeJoel SingerLaura Scott

Elixir PharmaceuticalsAlan Watson

The Progeria Research FoundationLeslie Gordon

NY State Institute for Basic Research

in Developmental DisabilitiesTed Brown

Coriell Cell Repositories

Children's Hospital OaklandPieter de JongYuko YoshinagaKazutoyo Osoegawa

University of Michigan / Department of Human GeneticsThomas GloverMichael GlynnSandra DurkinTony Csoka

Injection efficiency

Construct # Pups PCR screen- transgene positive (Fo)

tet 77 (wt) 61 25

tet75 (G608G) 59 29

BAC simple G608G

39 8

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

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

CT

GA

607 608 609