Stably transfected common fragile site sequences exhibit instability at ectopic sites.

Common fragile sites (CFSs) are loci that are especially prone to forming gaps and breaks on metaphase chromosomes under conditions of replication stress. Although much has been learned about the cellular responses to gaps and breaks at CFSs, less is known about what makes these sites inherently unstable. CFS sequences are highly conserved in mammalian evolution and contain a number of sequence motifs that are hypothesized to contribute to their instability. To examine the role of CFS sequences in chromosome breakage, we stably transfected two BACs containing FRA3B sequences and two nonCFS control BACs containing similar sequence content into HCT116 cells and isolated cell clones with BACs integrated at ectopic sites. Integrated BACs were present at just a few to several hundred contiguous copies. Cell clones containing integrated FRA3B BACs showed a significant, three to sevenfold increase in aphidicolin-induced gaps and breaks at the integration site as compared to control BACs. Furthermore, many FRA3B integration sites displayed additional chromosome rearrangements associated with CFS instability. Clones were examined for replication timing and it was found that the integrated FRA3B sequences were not dependent on late replication for their fragility. This is the first direct evidence in human cells that introduction of CFS sequences into ectopic nonfragile loci is sufficient to recapitulate the instability found at CFSs. These data support the hypothesis that sequences at CFSs are inherently unstable, and are a major factor in the formation of replication stress induced gaps and breaks at CFSs.

Incomplete processing of mutant lamin A in Hutchinson-Gilford progeria leads to nuclear abnormalities, which are reversed by farnesyltransferase inhibition.

Hutchinson-Gilford progeria syndrome (HGPS) is typically caused by mutations in codon 608 (G608G) of the LMNA gene, which activates a cryptic splice site resulting in the in-frame loss of 150 nucleotides from the lamin A message. The deleted region includes a protein cleavage site that normally removes 15 amino acids, including a CAAX box farnesylation site, from the lamin A protein. We investigated the processing of the C-terminus of the mutant protein, 'progerin', and found that it does not undergo cleavage and, indeed, remains farnesylated. The retention of the farnesyl group may have numerous consequences, as farnesyl groups increase lipophilicity and are involved in membrane association and in protein interactions, and is likely to be an important factor in the HGPS phenotype. To further investigate this, we studied the effects of farnesylation inhibition on nuclear phenotypes in cells expressing normal and mutant lamin A. Expression of a GFP-progerin fusion protein in normal fibroblasts caused a high incidence of nuclear abnormalities, as was also seen in HGPS fibroblasts, and resulted in abnormal nuclear localization of GFP-progerin in comparison with the localization pattern of GFP-lamin A. Expression of a GFP-lamin A fusion containing a mutation preventing the final cleavage step, causing the protein to remain farnesylated, displayed identical localization patterns and nuclear abnormalities as in HGPS cells and in cells expressing GFP-progerin. Exposure to a farnesyltransferase inhibitor (FTI), PD169541, caused a significant improvement in the nuclear morphology of cells expressing GFP-progerin and in HGPS cells. These results implicate the abnormal farnesylation of progerin in the cellular phenotype in HGPS cells and suggest that FTIs may represent a therapeutic option for patients with HGPS.

Mutation of the FOXC2 gene in familial distichiasis.

OBJECTIVE: To examine the FOXC2 gene in a family with hereditary distichiasis. BACKGROUND: Distichiasis, ie, a second row of eyelashes arising from the meibomian glands of the eyelids, can be inherited either alone (Online Mendelian Inheritance in Man [OMIM] no. 126300) or, more commonly, as part of the lymphedema-distichiasis (LD) syndrome (OMIM no. 153400). More than 45 families with mutations in the FOXC2 gene and LD have been described. Both lymphedema and distichiasis are highly penetrant. Distichiasis without lymphedema is not commonly seen. METHODS: We examined three generations of a family (N = nine members) with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was polymerase chain reaction--amplified from genomic DNA from all family members and examined for mutations. RESULTS: Clinical examination showed distichiasis of all four lids in two affected family members across two generations. There were no other consistent ophthalmologic abnormalities in the family. A cytosine-to-adenine transversion was identified in DNA from affected study participants at nucleotide position 1076, which would be predicted to cause truncation of the protein at codon 359. This change was not observed in any of the nine unaffected family members participating. CONCLUSIONS: This finding suggests that hereditary distichiasis and LD may not be separate genetic disorders but different phenotypic expressions of the same underlying disorder. Ophthalmologists should be aware that LD may present as distichiasis alone and counsel and refer their patients appropriately.

Recurrent de novo point mutations in lamin A cause Hutchinson-Gilford progeria syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by features reminiscent of marked premature ageing. Here, we present evidence of mutations in lamin A (LMNA) as the cause of this disorder. The HGPS gene was initially localized to chromosome 1q by observing two cases of uniparental isodisomy of 1q-the inheritance of both copies of this material from one parent-and one case with a 6-megabase paternal interstitial deletion. Sequencing of LMNA, located in this interval and previously implicated in several other heritable disorders, revealed that 18 out of 20 classical cases of HGPS harboured an identical de novo (that is, newly arisen and not inherited) single-base substitution, G608G(GGC > GGT), within exon 11. One additional case was identified with a different substitution within the same codon. Both of these mutations result in activation of a cryptic splice site within exon 11, resulting in production of a protein product that deletes 50 amino acids near the carboxy terminus. Immunofluorescence of HGPS fibroblasts with antibodies directed against lamin A revealed that many cells show visible abnormalities of the nuclear membrane. The discovery of the molecular basis of this disease may shed light on the general phenomenon of human ageing.