LMNA GENE

GENE THERAPY

REFERENCES

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CONCLUSION

INTRODUCTIONHutchinson-Gilford Progeria Syndrome, an egregious laminopathy, develops in 1 in 4 million infants between ages 12 and 24 months. A single de novo autosomal dominant mutation in the LMNA gene can lead to pleiotropic defects in array of different tissues, all stemming from nuclear-membrane buckling in somatic cells. Affected children suffer accelerated aging, cardiovascular defects, atherosclerosis, sclerotic skin, joint contractures, osteodysplasia, osteolysis, pathologic fractures, alopecia, growth retardation, micrognathia, and a dearth of subcutaneous fat. Patients are not cognitively impaired and remain mentally youthful as their bodies age. They most often die from myocardial infarction or stroke around thirteen years.

Figure 1. Phenotypic effects of HGPS 12

A. Picture of HGPS child manifesting classic signs of an undersize jaw, alopecia, lack of subcutaneous fat, and accelerated age.

B. Picture of normal nuclear lamina. C. Picture of “progerin-laden” nucleus

with characteristic abnormal morphology.

1A 1B

1C

PCB 3063, University of Florida, Gainesville, FL

MUTATIONS

PROTEINThe LMNA gene produces a polypeptide that requires post-translational processing to produce the mature lamin A protein, which functions as a nuclear protein scaffold significant to the integrity of the nuclear structure. In a study conducted by De Sandre-Giovannoli et al.2 on classical HGPS, a reverse transcriptase PCR isolated a normal transcript and a truncated mRNA transcript from lymphocytes derived from an affected child. The truncated transcript translated to a mutated form of pre-lamin A with an internal deletion of 50 amino acids, including the Zmpste24 cleavage site, due to activation of a cryptic splicing site. Thus, as seen in (Figure 6), the final cleavage of the 18 codons at the C terminus of the pre-lamin polypeptide cannot occur. Instead the resultant protein, progerin remains farnesylated (with a hydrocarbon at CAAX motif) and accumulates in the nuclear periphery.4 Progerin is then able intercalate into the nuclear membrane and dimerize with normal lamin A to form a protein complex that disrupts the intended protein scaffolding function; this results in the abnormal nuclear morphology characteristic of HGPS.1

Figure 5 . Significance of K542N position. K542N mutation (yellow) results in an AA substitution of an uncharged residue in the place of a charged residue within the DNA binding domain, which is largely positively charged (blue). http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846822/

Figure 6. Contrasting post-transcriptional processing. The mutant pre-lamin A has a 50AA deletion and subsequently lacks the final cleavage step that is characteristic to normal mature lamin A production prior to incorporation into the nuclear lamina. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2846822/

MODES OF INHERITANCE

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Atypical HGPS was observed in a study performed by Plasilova et al.11 on a consanguineous Indian family that supported a recessive mode of inheritance. The heterozygous offspring exhibited the affected phenotype (Figure 4).The investigators used genome wide linkage analysis to restrict the gene locus to 1p13.3–1q23.3, and additional microsatellite markers for further specificity. LMNA mutation analysis was used to confirm a G to C transversion that resulted in a missense mutation; in this K542N, a charged amino acid, lysine, was replaced by uncharged asparagine within the DNA binding domain of the protein (Figure 5).

Figure 4. Pedigree of four generations of an Indian family.11

Illustrates recessive inheritance. Consanguineous parents, I-1 and I-2, did not exhibit the disorder, and generated 5 heterozygous affected and 2 healthy offspring.

Classical HGPS is primarily inherited in a de novo dominant autosomal manner with a G608G mutation; despite this, affected individuals usually die before reaching reproductive age. The de novo nature of this mutation is unsurprising given that the cysteine of the CpG dinucleotide is easily methylated and deaminated to produce TpG.4

1. Capell, B.C., Erdos, M.R., Madigan, J.P., Fiordalisi, J.J., Varga, R., Conneely, K.N., Gordons, L.B., Der, C.J., Cox, A.D., Collins,

F.S. (2005). Proc. Natl. Acad. Sci. USA. 102, 12879-12884.

2. Csoka, A.B., Cao, H., Sammak, P.J., Caonstantinescu, D., Schatten, G.P., Hegele, R.A. (2004). Novel lamin A/C gene (LMNA)

mutations in atypical progeroid syndromes. J. Med. Genet. 41, 304-308.

3. De Sandre-Giovannoli, A., Bernard, R., Cau, P., Navarro, C., Amiel, J., Boccaccio, S.L., Stewart, C., Munnich, A., Le Merrer,

M., Lévy, N. (2003). Lamin A Truncation in Hutchinson-Gilford Progeria. Science. 300, 2055.

4. Eriksson, M., Brown, W.T., Gordon, L.B., Glynns, M.W., Singer, J., Scott, L., Erdos, M.R., Robbins, C.M., Moses, T.Y., Berglund,

P., et al., (2003). Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria

syndrome. Nature. 423, 293-298.

5. Garg, A., Subramanyam, L., Agarwal, A.K., Simha, V., Levine, B., D’ Apice, M.R., Novelli, G., Crow, Y. (2009). Atypical

Progeroid Syndrome due to Heterozygous Missense LMNA Mutations. J. Clin. Endocrinol. Metlab. 94, 4971-

4983.

6. Gordon, L.B. (2011). Hutchinson-Gilford Progeria Syndrome.

http://www.ncbi.nlm.nih.gov/books/NBK1121/#hgps.Management

7. Gordon, L.B., McCarten, K.M., Giobboe-Hurder, A., Machan, J.T., Campbell, S.E., Berns, S.D., Kieran, M.W. (2007). Disease

Progression in Hutchinson-Gilford Progeria Syndrome: Impact on Growth and Development. Pediatrics. 120,

824-833.

8. Glynn, M.W., Glover, T.W. (2005). Incomplete processing of mutant lamin A in Hutchinson-Gilford progeria leads to

nuclear abnormalities, which are reversed by farnesyltransferase inhibition. Human Molecular Genetics. 14,

2959-2969.

9. Marji, J., O’Donoghue, S.I., McClintock, D., Satagopasm, V.P., Schneider, R., Ratner, D., Womran, H.J., Gordon, L.B., Djabali,

K. Defective Lamin A-Rb Signling in Hutchinson-Gilford Progeria Syndrome and Reversal by

Farnesyltransferase Inhibition. PLoS Biol. 5, 1-14

10. McKusick, V.A. (2010). Hutchinson-Gilford Progeria Syndrome; HGPS.

http://www.ncbi.nlm.nih.gov/omim/176670#BiochemicalFeatures-176670.

11. Plasilova, M., Chattopadhyay, C., Pal, P., Schaub, N.A., Buechner, S.A., Mueller, H., Miny, P., Ghosh, A., Heinimann, K.

(2004). Homozygous missense mutation in the lamin A/C gene causes autosomal recessive Hutchinson-

Gilford progeria syndrome. 41, 609-614.

12. Raska, I. (2010). Importance of molecular cell biology investigations in human medicine in the story of the Hutchinson-

Gilford progeria syndrome. Interdisc Toxicol. 3, 89-93.

Scaffidi, P., Gordon, L., Misteli, T. (2005). The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. PLoS Biol. 3,

1855-1859.

13. Wydner, K.L., McNeil, J.A., Lin, F., Worman, H.J., Lawrence, J.B. (1996). Chromosomal Assignment of Human Nuclear

Envelope Protein Genes LMNA, LMNB1, and LBR by FluorescenceinsityHybridization. Genomics. 32, 474-

478.

14. Yang, S.H., Chang, A.Y., Ren, S., Wang, Y., Andres, D.A., Spielmann, H.P., Fong, L.G., Young, S.G. (2011). Absence of

progeria-like disease phenotypes in knock-in mice expressing a non-fanesylated version of progerin. Human

Molecular Genetics. 20, 436-444.

•Classical HPGS is most commonly caused by a c.1824C>T mutation. This C>T transition results in a silent Gly1824Gly mutation within the pre-lamin polypeptide.

•Despite the synonymous nature of this substitution, the mutation results in an unstable form of lamin A which generates a cryptic splice site that translates to an in-frame deletion of 150 nucleotides or 50 amino acids.

•The LMNA gene actually codes for both lamin A and lamin C, but the mutation only affects the structure of lamin A because exon 11 of LMNA is not present in lamin C mRNA.2

Eriksson et al.4 was able to isolate the c.1824C>T mutation. Samples from 23 people diagnosed with classical HGPS underwent PCR amplification followed by direct sequencing for all of the exons of the LMNA gene. The resulting sequences revealed that a significant 18 people out of 23 people were found to be heterozygous for the c.1824C>T mutation.

Because HGPS’s effect is not limited to specific tissue, but is instead universal to somatic cells,

there is no way to selectively repair the deleterious mutations throughout the body.

• The most promising treatment aims to reverse the HGPS-cell phenotype via farnesyl

transferase inhibitors (FTIs)

• FTIs bind to the CSIM sequence of pre-lamin A, blocking farnesyl transferase’s target and

preventing farnesyl addition.

• Lacking the bulky substituent, progerin remains free from the INM.

• FTIs are inevitably toxic as many proteins require farnesylation to maintain proper

functioning. Thus, FTIs are capable of disrupting normal cellular processes.

Figure 7. Immunofluorescence staining of

fibroblasts.1 The three pictures depict a normal

skin cell (far left), an HGPS skin cell (middle), and an

FTI-treated HGPS skin cell (far right).  Notice the

near-restoration of the FTI-treated nuclear lamina’s

spherical shape from the collapsed “progerin-laden”

HGPS cell.

Although rare, HGPS remains a great concern for its array of debilitating effects. Even more

frustrating perhaps is its resistance to therapies. Despite knowing its exact location, 1q21.2, its

somatic virulence eludes direct combat, relegating most medical interventions to high-calorie diets,

careful playing with other children, and persistence. The most promising treatment, FTIs, offer the

hope of rescuing the nuclear lamina from distortion in affected cells, but, again, at the risk of

compromising other, healthy protein and cellular processes in the body. Nevertheless, HGPS offers

intriguing insight into the massive epigenetic consequences of a single transition, swapping a

cysteine for a thymine.

Figure 2. LMNA gene position. mapped the LMNA gene to 1q21.2–q21.3, with emphasis on 1q21.3 as indicated by the black triangle. http://www.genecards.org/cgi-bin/carddisp.pl?gene=LMNA

The mapping of the LMNA gene, (Figure 2) was done by fluorescence in situ hybridization with a

DAPI counter-stain. Thus, specific assignment was made based upon location or distance from

DAPI bands and G-bands.14

Figure 3 . Table of HPGS mutations. c.1824C>T

represents the most common classical mutation for HGPS.

Atypical mutations are significantly more rare, but,are also

presented. They can result in amino acid substitutions as

well as alternative splice cites.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1735754/pdf/v

041p00e67.pdf

The classic mutation of HGPS has a low incidence of 1 in 18 million (Csoka, J Med Genet 2004). It is a de novo mutation; the mutation is in LMNA codon 608 at exon 11 of chromosome 1q of cytosine to thiamine at position 1824 (Csoka, J Med Genet 2004). The mutation

results in an unstable form of Lamin A which generates a cryptic site within the precursor mRNA for lamin A. The LMNA gene codes for both lamin A and lamin C, but the mutation only affects the structure of lamin A because exon 11 of LMNA is not present in lamin C

mRNA m (Csoka, J Med Genet 2004). The result is a silent gene mutation at codon 608 of Gly608Gly to Gly608Gly which results in an in-frame deletion of 150 nucleotides and 50 amino acids including sites required to process prelamin A to mature lamin A protein

The atypical mutation of HGPS can arise through a mutation on chromosome 1q at position 1822 of a Guanine to Adenine, a mutation on chromosome 1q at position 1921 of a Guanine to Adenine, a mild mutation: 35 amino acid deletion, a mutation at E145K, and a

joint mutation of R471C and R527C (Csoka, J Med Genet 2004).

The results of these mutations leads to synthesis of a mutant form of prelamin A, called, progerin, cannot be processed by mature lamin A. Synthesis of progerin, which lacks 50 amino acids near the C terminus, leads to misshapen nuclei in cultured cells and severe

disease phenotypes in affected patients. Retention of farnesyl group causes progerin to become permanently anchored in the nuclear membrane and unable to be released. The central rod domain of progerin then allows dimerization with mature nonfarnesylated LA

and assembly into a multiprotein complex, resulting in dominant-negative disruption of the nuclear scaffolding and underlying heterochromatin and leading to the characteristic nuclear blebbing seen in HGPS (Capell, PNAS 2005).

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1. De Sandre-Giovannoli, A., Bernard, R., Cau, P., Navarro, C., Amiel, J., Boccaccio, S.L., Stewart, C., Munnich, A., Le Merrer, M., Lévy, N. (2003). Lamin A Truncation in Hutchinson-Gilford Progeria. Science. 300, 2055.2. Domingo, D.L., Trujillo, M.I., Council, S.E., Merideth, M.A., Gordon, L.B., Wu, T., Introne, W.J., Gahl, W.A., Hart, T.C. (2009). Hutchinson-Gilford progeria syndrome: Oral and craniofacial phenotypes. Oral Dis. 15, 187-195.3. Eriksson, M., Brown, W.T., Gordon, L.B., Glynns, M.W., Singer, J., Scott, L., Erdos, M.R., Robbins, C.M., Moses, T.Y., Berglund, P., et al., (2003). Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome. Nature. 423, 293-298. 4. Garg, A., Subramanyam, L., Agarwal, A.K., Simha, V., Levine, B., D’ Apice, M.R., Novelli, G., Crow, Y. (2009). Atypical Progeroid Syndrome due to Heterozygous Missense LMNA Mutations. J. Clin. Endocrinol. Metlab. 94, 4971-4983. 5. Gordon, L.B. (2011). Hutchinson-Gilford Progeria Syndrome. http://www.ncbi.nlm.nih.gov/books/NBK1121/#hgps.Management6. Gordon, L.B., McCarten, K.M., Giobboe-Hurder, A., Machan, J.T., Campbell, S.E., Berns, S.D., Kieran, M.W. (2007). Disease Progression in Hutchinson-Gilford Progeria Syndrome: Impact on Growth and Development. Pediatrics. 120, 824-833.7. Glynn, M.W., Glover, T.W. (2005). Incomplete processing of mutant lamin A in Hutchinson-Gilford progeria leads to nuclear abnormalities, which are reversed by farnesyltransferase inhibition. Human Molecular Genetics. 14, 2959-2969. 8.Marji, J., O’Donoghue, S.I., McClintock, D., Satagopasm, V.P., Schneider, R., Ratner, D., Womran, H.J., Gordon, L.B., Djabali, K. Defective Lamin A-Rb Signling in Hutchinson-Gilford Progeria Syndrome and Reversal by Farnesyltransferase Inhibition. PLoS Biol. 5, 1-149. McKusick, V.A. (2010). Hutchinson-Gilford Progeria Syndrome; HGPS. http://www.ncbi.nlm.nih.gov/omim/176670#BiochemicalFeatures-176670.10. Plasilova, M., Chattopadhyay, C., Pal, P., Schaub, N.A., Buechner, S.A., Mueller, H., Miny, P., Ghosh, A., Heinimann, K. (2004). Homozygous missense mutation in the lamin A/C gene causes autosomal recessive Hutchinson-Gilford progeria syndrome. 41, 609-614.11. Raska, I. (2010). Importance of molecular cell biology investigations in human medicine in the story of the Hutchinson-Gilford progeria syndrome. Interdisc Toxicol. 3, 89-93.12. Russo-Menna, I., Arancibias, C. (2010). The Hutchinson-Gilford Progeria Syndrome: a case report. Minerva Anestesiologica. 76, 151-154.13. Scaffidi, P., Gordon, L., Misteli, T. (2005). The Cell Nucleus and Aging: Tantalizing Clues and Hopeful Promises. PLoS Biol. 3, 1855-1859.14. Yang, S.H., Chang, A.Y., Ren, S., Wang, Y., Andres, D.A., Spielmann, H.P., Fong, L.G., Young, S.G. (2011). Absence of progeria-like disease phenotypes in knock-in mice expressing a non-fanesylated version of progerin. Human Molecular Genetics. 20, 436-444.15.Yang, S.H., Qiao, X., Farber, E., Chang, S.Y., Fong, L.G., Young, SG. (2007). Eliminating the Synthesis of Mature Lamin A Reduces Disease Phenotypes in Mice Carrying a Hutchinson-Gilford Progeria Syndrome Allele. J. Bio. Chem. 283, 7094-7099.

The LMNA gene was mapped to 1q21.2–q21.3, with positional emphasis on 1q21.3, as determined by fluorescence in situ hybridization with a DAPI counter-stain.

The majority of patients with HGPS have de novo heterozygous dominant mutations in the LMNA gene. Presumably, patients with the disorder do not survive long enough to reproduce (Eriksson et al., 2003; Cao and Hegele, 2003).