Lamin A Truncation in Hutchinson-Gilford Progeria

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Science  27 Jun 2003:
Vol. 300, Issue 5628, pp. 2055
DOI: 10.1126/science.1084125

Little is known about the pathophysiology of human senescence. Hutchinson-Gilford progeria syndrome (HGPS) is an exceedingly rare but typical progeria, clinically characterized by postnatal growth retardation, midface hypoplasia, micrognathia, premature atherosclerosis, absence of subcutaneous fat, alopecia, and generalized osteodysplasia with osteolysis and pathologic fractures. The median age at death is 13.4 years, usually due to coronary artery disease (1). Dominant inheritance is likely, recessive cases probably being the result of germinal mosaicism (1).

Lamin A/C proteins, encoded by the LMNA gene, are major, ubiquitous components of nuclear laminae (2). A wide spectrum of human disorders is ascribed to mutations at the LMNA locus (3). The mandibulo-acral dysplasia phenotype, resulting from a LMNA founder mutation, shares several features with HGPS, although life expectancy is not shortened (4). Thus LMNA is a strong candidate gene for HGPS.

In a genomic and transcriptional analysis of LMNA in children affected with HGPS (Fig. 1A), two patients had a heterozygous C to T transition at nucleotide 1824 (C1824 to T1824) in exon 11 of the coding sequence (5). This substitution had no postulated effect on the translated amino acid (Gly608), but because it was found neither on 300 control chromosomes nor in the parents, we investigated whether it affected transcript splicing. On one patient's lymphocytes, reverse transcription polymerase chain reaction amplification of messenger RNAs using primers located in exons 9 and 12 found two fragments, the larger corresponding to the expected transcript size, as in controls (fig. S1). The shorter transcript had a 150–base pair (bp) deletion, removing the 3′ half of exon 11. The deletion started at G1819 because a cryptic donor splicing site was used at position 1819 to 1820. Fifty amino acids were predicted to be removed from Lamin A tail, leaving Lamin C unmodified (Fig. 1B). The other transcript had a normal coding sequence length, although it carried a T at position 1824. To determine on which allele the deletion occurred, we further analyzed the genomic and transcribed sequences upstream of the deletion to identify informative polymorphisms. Both transcripts were produced by the same allele (T at position 1824) (fig. S1). The consequences of the mutation are described as follows: [r.1824c>t + r.1819_1968del], i.e., the c.1824C>T change causes the appearance of one RNA molecule carrying the variation and another containing a deletion of nucleotides 1819 to 1968. Thus, in patients Lamin A may not be transcribed from the normal allele (C at position 1824). The mutation would then result in truncated and normal mRNAs in cis, whereas, in trans, it would inhibit transcriptional processing of the normal allele, acting as a dominant negative mutation.

Fig. 1.

(A) HGPS affecting a 6-year-old female. (B) Representation of the LMNA gene and Lamin A protein, correlated by blue (globular domains) and red (rod domain) colors. The deleted LMNA transcript junction sequence is shown. The 150-bp deletion extends from G1819 to exon 11 end (black box) and is indicated by a black bar in Lamin A tail. CaaX, cysteine-aliphatic-aliphatic-any amino acid. (C) Lymphocyte nuclei from controls: (a) Detection of Lamin A/C and (c) Lamin A. (b and d) DAPI (4′,6′-diamidino-2-phenylindole) staining. (D) Lymphocyte nuclei from the patient in (A). (a) Detection of Lamin A/C; (c) Absence of Lamin A. (b and d) DAPI staining. (e) Lamin B1 localizes both at nuclear envelope and nucleoplasm. (f) May-Grünwald-Giemsa staining shows nuclear deformities and cytoplasmic vacuoles. Width of fluorescent images, 80 μm. Width of brightfield images, 120 μm.

Immunocytochemical analyses with specific antibodies to Lamins A/C, A, and B1 were performed (5). HGPS lymphocytes, which were larger than control ones, exhibited large cytoplasmic vacuoles and abnormal mitotic figures. Most cells had strikingly altered nuclear sizes and shapes, with envelope interruptions accompanied by chromatin extrusion (Fig. 1D). Lamin A was only detected in 10 to 20% of HGPS lymphocytes, whereas Lamin A/C staining indicated the presence of only Lamin C in most cells (Fig. 1D, a and c). Moreover, Lamin B1 was delocalized to the nucleoplasm, likely due to the loss of Lamin A (Fig. 1D, e). These alterations were significant compared with control cells (Fig. 1C). Western blotting showed that the patient expressed 25% of normal Lamin A levels, whereas no truncated form was detected (fig. S1) (5). Thus, HGPS appears to represent a novel laminopathy, caused by a single heterozygous splicing mutation in the LMNA gene, leading to a major loss of Lamin A expression, intimately associated to nuclear alterations.

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Materials and Methods

Fig. S1

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