In this study we identified two novel mutations and a third previously described mutation in ZMPSTE24
that are associated with restrictive dermopathy. Importantly, we also show that RD is a simple autosomal recessive disorder. Though ZMPSTE24
had already been implicated in RD, only one heterozygous mutation was identified, and it was also present in unaffected family members, leading to the conclusion that RD was a digenic disorder (Navarro et al., 2004
). Rather than there being a digenic mode of inheritance, we favor the possibility that there exist ZMPSTE24
mutations that are difficult to detect by conventional PCR-based methods. This is perhaps the most straightforward explanation for how there could be an absence of mature lamin A even with an apparently wild-type copy of ZMPSTE24
. Such mutations could lie in the gene’s promoter, or could involve segmental deletions that are complemented--only as far as PCR is concerned--by normal regions of the other allele. Indeed, we could not find any mutations in a pair of affected fraternal twins from Finland, though SNP genotyping showed that the twins share genotypes on a segment of chromosome 1 containing the ZMPSTE24
locus (data not shown).
All mutations identified thus far to cause RD are single base duplications that result in mRNA frameshifts, which likely prevent production of a functional protein. However, one missense ZMPSTE24
mutation (c.1018T>C; p.Trp340Arg) has been found, in a Belgian patient with severe mandibuloacral dysplasia with type B lipodystrophy (MADB—MIM 608612) (Agarwal et al., 2003
). This patient was actually compound heterozygous; the other allele bore the same c.1085dupT inactivating mutation that Navarro et al. 2004
and we have subsequently found to be associated with RD. The mutant protein carrying the p.Trp340Arg substitution is partially active (Agarwal et al., 2003
), and this easily explains the fact that MADB is a less severe disease than RD.
Accumulation of prelamin A has been reported in Zmpste24
knockout mice (Bergo et al., 2002
; Pendas et al., 2002
) and in cultured cells from human patients with Hutchinson-Gilford progeria syndrome (HGPS—MIM 176670) (Goldman et al., 2004
); its presence has also been hypothesized but not proven in other human laminopathies (Agarwal et al., 2003
; Navarro et al., 2004
). Ours is the first demonstration of accumulation of prelamin A in affected human tissue in situ (Fig. 4A), suggesting that it is not merely a consequence of cell culture. Accumulation of prelamin A has recently been shown to be toxic; disease phenotypes in Zmpste24
−/− mice are “rescued” by only a 50% reduction in the amount of prelamin A, via knockout of one Lmna
allele (Fong et al., 2004
). The presence of large amounts of prelamin A may also account for the increased severity of RD as compared to laminopathies with either lamin A mutations or missense mutation in ZMPSTE24
. Given the lack of detectable prelamin A in normal cells, it is possible that a simple immunofluorescence assay for prelamin A in fetal cell nuclei could serve as a prenatal test for RD and perhaps other laminopathies.
We found lamins A and C clustered and distributed in aggregates in the nuclei of RD patient fibroblasts, whereas normal fibroblast nuclei showed a homogeneous and smooth, even distribution, concentrated at the nuclear periphery. Abnormal distribution of lamins A and C in foci, aggregations, and honeycombs has been reported in a number of human patients carrying lamin A/C gene mutations (Capanni et al., 2003
; Caux et al., 2003
; Muchir et al., 2004
; Novelli et al., 2002
) as well as in cells transfected with lamin A/C mutants (Holt et al., 2003
; Ostlund et al., 2001
The mechanism whereby mutations in LMNA
that result in aberrant lamin A processing cause disease has not been determined. Several lines of evidence suggest that it is likely to be a combination of impaired nuclear stability and the resulting nuclear deformations (Lammerding et al., 2004
) causing secondary alterations in heterochromatin localization and significant changes in gene expression (Nikolova et al., 2004
). (For a review of these hypotheses, see reference (Worman and Courvalin, 2004
).) In addition, lamin A binds directly or indirectly to a number of nuclear proteins, including emerin (Lee et al., 2001
; Sakaki et al., 2001
) and nesprin (Mislow et al., 2002
), and attach them to the nuclear envelope. Lamin A also binds to transcription factors such as MOK2 (Dreuillet et al., 2002
), retinoblastoma (RB) (Mancini et al., 1994
), and SREBP1 (Lloyd et al., 2002
). (See (Zastrow et al., 2004
) for a review of lamin-binding proteins.) Mislocalization of lamin A, as we and others have observed, is likely indicative that its binding partners are also mislocalized and not bound to the nuclear envelope, which may have profound secondary effects on cells.
Interestingly, the neonatally lethal wrinkle free phenotype of Slc27a4
−/− mice that we and others have reported (Herrmann et al., 2003
; Moulson et al., 2003
) appears to be much more similar to the human RD phenotype than that exhibited by Zmpste24
−/− mice, which survive for several months. This may be coincidence, but the possibility exists that there is some special mechanistic relationship in humans between nuclear architecture and expression of genes involved in fatty acid homeostasis. Indeed, one of the features of the diverse laminopathies that are less severe than RD is lipodystrophy.
Genetic and allelic heterogeneity appear to explain in part the phenotypic differences among the related laminopathies Hutchinson-Gilford Progeria Syndrome (HGPS-MIM 176670), MAD, and most recently, RD. Navarro et al.
included in their study two patients several months old with a skin disease less severe than typical RD, and they found one novel and one previously reported mutation in the LMNA
gene. These are both likely to be sporadic, heterozygous, dominant negative mutations. The latter mutation had been found previously in several patients with HGPS. There is clearly a marked difference in disease severity between ZMPSTE24
-based neonatally lethal RD and LMNA
-based progeria. To avoid confusion, we propose that the descriptor “restrictive dermopathy” be reserved for individuals with the severe, neonatally lethal disease described by Witt (Witt et al., 1986
). Children with some RD-like features who survive well past the neonatal period are more likely to manifest features of progeria or MAD and should be described as such. The ability to screen for ZMPSTE24
mutations should make this distinction relatively straightforward.