Increased expression of the Hutchinson-Gilford progeria syndrome truncated lamin A transcript during cell aging

Abstract

Most cases of the segmental progeroid syndrome, Hutchinson-Gilford progeria syndrome (HGPS), are caused by a de novo dominant mutation within a single codon of the LMNA gene. This mutation leads to the increased usage of an internal splice site that generates an alternative lamin A transcript with an internal deletion of 150 nucleotides, called lamin A Delta 150. The LMNA gene encodes two major proteins of the inner nuclear lamina, lamins A and C, but not much is known about their expression levels. Determination of the overall expression levels of the LMNA gene transcripts is an important step to further the understanding of the HGPS. In this study, we have performed absolute quantification of the lamins A, C and A Delta 150 transcripts in primary dermal fibroblasts from HGPS patients and unaffected age-matched and parent controls. We show that the lamin A Delta 150 transcript is present in unaffected controls but its expression is >160-fold lower than that in samples from HGPS patients. Analysis of transcript expression during in vitro aging shows that although the levels of lamin A and lamin C transcripts remain unchanged, the lamin A Delta 150 transcript increases in late passage cells from HGPS patients and parental controls. This study provides a new method for LMNA transcript analysis and insights into the expression of the LMNA gene in HGPS and normal cells.

Figure 1

LMNA gene transcript quantification. Amplicons for lamins A, AΔ150 and C transcript quantification located in the 3′ end of the LMNA gene (exons 9–12). Blue arrows and orange bars show the location of primers and TaqMan probes, respectively. The reverse primers for the lamins A and AΔ150 assays, and the forward primer for the lamin C assay covered exon–exon borders. The part of exon 10 specific for lamin C is marked in blue. The 150 nucleotide region of exon 11, which is deleted because of alternative splicing in the lamin AΔ150 transcript is marked in yellow. Not to scale.

Figure 2

Absolute quantification of lamin AΔ150, A and C transcripts in primary fibroblast from HGPS patients and unaffected controls. Same data were plotted in (a) and (b), and results represent means ±SD. RNA was extracted from different passages of primary dermal fibroblasts from seven Hutchinson–Gilford progeria syndrome patients (HGPS, n=34), three unaffected age-matched (n=12), and four unaffected parent controls (n=24). Asterisk represent significant differences: ^*P<0.05, ^**P<0.01, and ^***P<0.001.

Figure 3

LMNA locus transcripts in early and late passage cells. Results represent means and ±SD. Asterisk represent significant differences: ^*P<0.05, ^**P<0.01.

Figure 4

A-type lamins in primary fibroblasts from HGPS and unaffected controls. (a) Western blot experiments with antibodies to lamins A (left panel), C (middle panel), and A/C (right panel). (b) RT-PCR screening with exon (ex) 7/8 or ex9/11, and ex12 primer showed fragments corresponding to lamins A (640 base pairs, bp) and AΔ150 (490 bp), or lamins AΔ10 (319 bp) and AΔ10, Δ150 (169 bp), respectively. RT-PCR with primers in ex1, 2, or 3, and ex10, resulted in fragments corresponding to lamin C (1612, 1350, and 1118 bp, respectively). Asterisk (^*) indicates that the primer was located in the region of exon 10 specific for lamin C. Negative control lanes contained PCR samples with genomic DNA, no RT, and no template controls (M, 100 bp DNA ladder (Invitrogen)). (c) Representative western blot with protein extracts from multiple passages of fibroblast cell cultures. (d) Changes in protein levels during passaging illustrated as the ratio of normalized band intensities for late and early passage protein extracts of the same culture. Results represent means ±SD.