Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome

Abstract

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by features reminiscent of marked premature ageing. Here, we present evidence of mutations in lamin A (LMNA) as the cause of this disorder. The HGPS gene was initially localized to chromosome 1q by observing two cases of uniparental isodisomy of 1q-the inheritance of both copies of this material from one parent-and one case with a 6-megabase paternal interstitial deletion. Sequencing of LMNA, located in this interval and previously implicated in several other heritable disorders, revealed that 18 out of 20 classical cases of HGPS harboured an identical de novo (that is, newly arisen and not inherited) single-base substitution, G608G(GGC > GGT), within exon 11. One additional case was identified with a different substitution within the same codon. Both of these mutations result in activation of a cryptic splice site within exon 11, resulting in production of a protein product that deletes 50 amino acids near the carboxy terminus. Immunofluorescence of HGPS fibroblasts with antibodies directed against lamin A revealed that many cells show visible abnormalities of the nuclear membrane. The discovery of the molecular basis of this disease may shed light on the general phenomenon of human ageing.

Figure 1

Localization of the HGPS gene to a 4.82-Mb interval on chromosome 1q. a, Uniparental isodisomy of chromosome 1q in fibroblasts from two patients with HGPS (filled symbols). A subset of markers and their genotypes are shown. NA, not available b, The boxed interval is the region in proband C8803 that has been inherited exclusively from the mother. c, FISH analysis of a metaphase spread from C8803 fibroblasts hybridized with BAC probes RP11-110J1 (green) and RP11-91G5 (red). d, 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.

Figure 2

Point mutations in exon 11 of LMNA cause HGPS. a, Sequence traces from a normal control and two HGPS patients. b, Hypothesis for activation of a cryptic splice donor site in exon 11. c, Demonstration of the abnormal splice product using RT–PCR, showing an abnormal product of 489 bp in the two HGPS probands due to activation of the cryptic splice site. Alternative lanes to the right lack reverse transcriptase. d, Western blot using a monoclonal antibody against lamin A/C. Lanes 1, 5, 8 and 9 are from AG03506, AG03344, AG11498 and AG01972; they 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.

Figure 3

Immunofluorescence on primary dermal fibroblasts from an unaffected mother and a child with classical HGPS, using antibody JOL2 against lamin A/C. Identical results were obtained with antibody XB10 (data not shown). a–d, Results from an unaffected mother, AG06299. e–h, Results from a classical HGPS patient, AG11498. a, e, Antibody against lamin A/C. b, f, DAPI staining showing location of the nuclei. c, g, Antibody staining mitochondria, showing distribution of the cytosol. d, h, Merged image of a–c (d) and e–g (h).