Construction of Minigenes of LMNA to Study Progeria




Dayi Li

Introduction

Progeria is a premature aging syndrome that was first described by Dr. Jonathan Hutchinson in 1886 and Dr. Hastings Gilford in 1897. The condition was later named Hutchinson–Gilford progeria syndrome (HGPS). The term progeria is often applied specifically in reference to HGPS[1]. As an extremely rare genetic disorder, HGPS resembles aspects of aging at a very early age. This early onset, acute disease will cause wrinkled skin, severe cardiovascular problems, kidney failure, loss of eyesight, and premature death. Those born with progeria only live to their mid-teens or early twenties. In this project, I generated minigenes of LMNA carrying progeria causing mutations. A better understanding of HGPS may reveal clues about the normal aging process.

HGPS is caused by mutations in the LMNA gene. The LMNA gene encodes for a structural protein which undergoes a series of processing steps before attaining its final form, called lamin A. Lamin A makes up the nuclear lamina to provide structural support to the nucleus. The protein is farnesylated in cytoplasm and is transported to the interior of the nucleus. The farnesyl group allows lamin A to attach temporarily to the nuclear rim and then is removed by a protease. If the farnesyl group cannot be removed, lamin A is permanently affixed to the nuclear rim. So the nuclear lamina is unable to provide the nuclear envelope with adequate structural support. Chromatin organization during mitosis is interrupted, leading to compromised cell division[2].

Most HGPS cases are caused by one of two dominant, de novo, point mutations p.G608G (GGC > GGT) and p.G608S(GGC > AGC). The mutation p.G608G does not change an amino acid. The mutation p.G608S results in a substitution of serine for glycine.

The study in 2003 looked further into the normal sequence surrounding codon 608. By comparing this region to the consensus splice donor sequence, they proposed that both of the observed HGPS mutations result in activation of a cryptic splice site within exon 11 (Figure 1)[3]. As a consequence, the terminal 150 bases of exon 11 are removed, resulting in the removal of 50 amino acids of the protein, producing a truncated protein (progerin or LaminAΔ50).

The purpose of this study was to generate minigenes of LMNA that carry the progeria causing mutations. Using the mutation-containing minigenes, we can study the impact of these mutations on the splicing of exon 11.

Materials and Methods

DNA extracted from two cell lines (HL-60 and U2OS) was used to amplify the exon 10 to exon 12 region of the wild-type LMNA gene. This amplified product was inserted into the pcDNA3.1/V5-His TOPO vector to generate the wild-type-containing construct.

Cells were transfected with this minigene construct. RNA was extracted, and RT-PCR was performed to examine plasmid derived transcript.

To generate minigenes with either the p.G608G (GGC > GGT) mutation or the p.G608S(GGC > AGC) mutation, site-directed mutagenesis of the wild-type-containing construct was performed.

Sequencing results of minigene constructs and transcript were verified using the NCBI’s database.

Results

DNA template was obtained from HL-60 and U20S cell lines. Primers were designed to amplify genomic region from the exon 10 to exon 12 of LMNA gene. The PCR products were analyzed in 1% agarose gel. The size showed in Figure 2 is 2.5kb as expected. Construct was sent out for sequencing. Sequencing results were verified with NCBI’s database.

Cells were transfected with wild-type-containing minigene. RNA was extracted. RT-PCR was performed to examine plasmid derived transcript. Primers were designed to amplify region from the vector to the beginning of exon 12. The RT-PCR product was analyzed in 1% agarose gel. The size showed in Figure 3 is 463bp as expected. RT-PCR product was purified and sent out for sequencing. Sequencing result confirmed that wild-type-containing minigene generated the expected transcript.

Using site-directed mutagenesis, minigenes that contain one of the two progeria causing mutations were generated. Sequencing results confirmed that these two minigene constructs carry desired mutations, respectively, as shown in Figure 4.

Discussion

In this study, I successfully amplified the genomic region from exon 10 to exon 12 of LMNA gene. Wild-type-containing construct was generated. Two progeria causing mutations constructs were generated by site-directed mutagenesis of the wild-type-containing construct, respectively. Meanwhile, transcript expression analysis showed that wild-type-containing minigene generated the expected transcript.

These three minigene constructs provide a valuable tool for researchers evaluating splicing patterns of progeria in vivo. Now we can study the impact of these mutations on the splicing of exon 11. Potential cis-regulatory elements and trans-regulatory elements that affect LMNA gene expression can be identified.

Further work may include examining the LMNA transcript of cells transfected with the mutation containing construct.

References

[1] Ramirez C L, Cadinanos J, Varela I, et al. Human progeroid syndromes, aging and cancer: new genetic and epigenetic insights into old questions[J]. Cellular and molecular life sciences, 2007, 64(2): 155-170.

[2] Cao K, Graziotto J J, Blair C D, et al. Rapamycin reverses cellular phenotypes and enhances mutant protein clearance in Hutchinson-Gilford progeria syndrome cells[J]. Science translational medicine, 2011, 3(89): 89ra58-89ra58.

[3] Eriksson M, Brown W T, Gordon L B, et al. Recurrent de novo point mutations in lamin A cause Hutchinson–Gilford progeria syndrome[J]. Nature, 2003, 423(6937): 293-298.

Abstract

As an extremely rare genetic disorder, Hutchinson–Gilford progeria syndrome(HGPS) resembles aspects of aging at a very early age. A better understanding of HGPS may reveal clues about the normal aging process.

In this project, LMNA wild-type-containing minigene construct was generated. Two progeria causing mutations constructs were generated by site-directed mutagenesis of the wild-type-containing construct, respectively. Meanwhile, transcript expression analysis showed that wild-type-containing minigene generated the expected transcript. As a valuable tool for evaluating alternating splicing patterns of progeria, now we can study the impact of these mutations on the splicing of exon 11 of LMNA.

Figures


Figure 1-Hypothesis for activation of a cryptic splice donor site in exon 11.


Figure 2-Successful amplification of the exon 10 to 12 region of LMNA. Amplicon is 2.5kb as expected.


Figure 3-Amplification of the plasmid derived transcript. Amplicon of transcript is 463bp as expected.


Figure 4-Using site-directed mutagenesis, minigenes that contain one of the two progeria causing mutations were generated. Sequencing results confirmed that these two minigene constructs carry desired mutations, respectively.


Full Paper

Acknowledgments

I would like to thank Dr. Rubin for his expert guidance through this project. I would also like to thank Anthony Evans, Catharina Grubaugh and Faaria Fasih for their patience and assistance. Thanks also go to my cohort for the encouragement.


This document was last modified 05/17/2017.
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