Molecular studies of Hutchinson-Gilford progeria syndrome

University dissertation from Stockholm : Karolinska Institutet, Biosciences and Nutrition

Abstract: Hutchinson-Gilford progeria syndrome (HGPS) is a very rare genetic disease, with an incidence of 1 in 4-8 million live births, that causes segmental premature aging in children. The children look normal at birth but begin to develop symptoms of disease within the first years of life. The symptoms include growth retardation, scleroderma, osteoporosis and atherosclerosis of the coronary and cerebrovascular arteries. Myocardial infarction or stroke is the most common causes of death at a median age of 13 years. The aims of this work includes: to increase the understanding of the molecular mechanisms underlying progeria, to see if there is any possibility of disease reversal and to develop a specific method for analyzing LMNA transcripts during normal and in vitro aging. For these purposes, we developed an inducible tissue-specific transgenic mouse model system that included a minigene of human LMNA with either the wild-type sequence or the most common HGPS mutation, 1824C>T, and assays for absolute quantification of the LMNA transcripts in HGPS patient material and controls of different ages. PAPER I: Animal models are crucial to increase understanding of the ongoing molecular process during disease, especially for rare and severe diseases like HGPS. To get a better understanding of the HGPS skin phenotype, we developed an inducible and tissue specific model system with keratin 5- targeted transgenic expression. Bitransgenic animals with the HGPS mutation have a progressive phenotype. The phenotype is first characterized by an intermediate stage with varying degrees of hyperplasia of the interfollicular epidermis, mis-expression of keratins 5 and 6 and increased proliferation. The end stage is seen later, with loss of subcutaneous fat and fibrosis of the dermis, similar abnormalities to those seen in the skin of HGPS patients. The severity of the disease phenotype correlates with the level of transgenic expression (higher expression gives more severe disease phenotype). Animals expressing the wild-type allele had a normal appearance of the skin. PAPER II: To examine if expression of progerin affects the expression of lamin B or the progress of the hair cycle, we first characterized the normal expression of lamin A/C and B in mouse skin cell types during hair cycling. Immunohistochemical staining of the whole back skin of FVB/NCrl wild-type mice showed strong expression of lamin A/C and B in the basal layer of the epidermis, the outer root sheath of the hair follicle and the dermal papilla during all stages of the hair cycle. Lower expression was seen in the suprabasal cells of the epidermis, in the hypodermis and the bulb of catagen follicles. Analyzing the different phases of the first postnatal hair cycle and the expression of lamin B in our mouse model of HGPS did not reveal any shifts in the hair cycle or in the expression of lamin B. PAPER III: To examine if progeria disease is reversible and learn more about the possibility of treatment for the children with progeria who are already manifesting the disease, we used our inducible transgenic mouse model of HGPS. After disease development, transgenic expression was suppressed and the animals were observed for reversal in disease phenotype. The external phenotype of hair loss and skin crusting improved after only 1 week of suppression and after 6 or 13 weeks the external skin phenotype looked completely normal in most of the animals. The lower weights of bitransgenic animals increased after transgenic suppression and followed the trend of the wild-type curve. The disease pathology seen in the skin of bitransgenic animals was almost indistinguishable from wild-type after 6 and 13 weeks of suppressed transgenic expression. This shows that the expression of the HGPS mutationdoes not cause irreversible damage, at least in these tissues, which gives promise for future treatment for this disease. PAPER IV: To characterize the expression levels of the LMNA locus transcripts in HGPS patients and age-matched and parental controls and during in vitro aging, we developed a method for absolute quantification using real-time RT-PCR. Lamin A, C and lamin Adelta150 transcripts were quantified in HGPS and normal cells of different ages. Our results showed that lamin C is the most highly expressed transcript from the LMNA locus. The lamin Adelta150 transcript was present in unaffected controls but at >160-fold lower levels than in HGPS patient cells. While the levels of lamin A and C remained unchanged during in vitro aging, the lamin Adelta150 transcript increased in late passage cells from both HGPS and parental controls, which suggests that similar mechanisms exist in HGPS and normal aging cells.

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