Progeroid laminopathy with restrictive dermopathy-like features caused by an isodisomic LMNA mutation p.R435C

Introduction

Laminopathies are a group of rare genetic diseases caused by mutations in genes encoding proteins of the nuclear lamina (primary laminopathies) or proteins interacting with lamina proteins (secondary laminopathies). A group of these laminopathies have been classified as progeroid syndromes that can be caused by mutations in LMNA or ZMPSTE24 [1]. LMNA is encoding lamin A and lamin C by alternative splicing, while ZMPSTE24 is encoding the zinc metalloproteinase ZMPSTE24, which is necessary for the processing of prelamin A to mature lamin A. Therefore LMNA mutations are classified as primary laminopathies whereas ZMPSTE24 mutations are included in the group of the secondary laminopathies. Progeroid syndromes mimic clinical and molecular features of aging. Apart from some atypical progeroid forms there are three major syndromes caused by LMNA or ZMPSTE24 mutations: Hutchinson Gilford progeria syndrome (HGPS), Mandibuloacral Dysplasia (MAD) and Restrictive Dermopathy (RD).

HGPS is most commonly caused by the heterozygous de novo lamin A mutation p.G608G [2-4], which activates a cryptic splice site and causes a deletion of 50 amino acids on the protein level including the C-terminal cleavage site for ZMPSTE24. The resulting farnesylated mutant lamin A, known as progerin, accumulates inside the nucleus. The affected children appear healthy at birth and, in the course of 1-2 years progressively develop a so-called progeroid phenotype, comprising extreme short stature, low body weight, early loss of hair, lipodystrophy, scleroderma, decreased joint mobility, osteolysis, and facial features resembling aging[2, 5]. Cardiovascular problems lead in most cases to death in the second decade.

MAD can be associated with either homozygous or compound heterozygous missense mutations in LMNA (MADA) or a combination of a nonsense and a missense mutation in ZMPSTE24 (MADB) [6-8]. MAD patients are characterized by postnatal growth retardation, craniofacial anomalies like mandibular hypoplasia (or osteolysis) and protruding mid-face as well as skeletal anomalies including progressive osteolysis of the terminal phalanges and clavicles. Skin changes like atrophy and speckled hyperpigmentation have been observed. Furthermore clinical features like displaced teeth, thin and brittle hair, short and wide fingernails as well as accumulation of fat in the neck and moderate lipodystrophy of the limbs are typical.

RD describes phenotypically a lethal neonatal genodermatosis characterized by tautness of the skin causing fetal akinesia and often premature delivery. Other clinical features include tightly adherent thin skin, prominent vessels, characteristic facial features, generalized joint contractures, dysplasia of clavicles and respiratory insufficiency. The clinical course of RD is fatal and, with respect to HGPS or MAD, more severe leading to neonatal death or death in early infancy [9]. LMNA-linked heterozygous RD mutations cause a skipping of exon 11 and lead to a deletion of 90 amino acids [7]. The resulting truncated protein cannot undergo full posttranslational maturation, similar to progerin, and is accumulated inside the nucleus. In ZMPSTE24-linked RD, homozygous or compound heterozygous nonsense mutations are causing the complete absence of the essential prelamin A processing enzyme ZMPSTE24 resulting in an extreme accumulation of normal length lamin A precursors.

A common feature of at least HGPS and RD is the accumulation of prelamin A or truncated forms of it. Pathogenic effects of prelamin A accumulation have been associated with altered chromatin organization and impaired DNA-damage response pathways, resulting in increased genomic instability, susceptibility to DNA-damaging agents and double strand breaks (dsb). Indeed such abnormalities are observed in patients with HGPS or RD as well as in cultured human and murine cells with aberrant lamin A processing pathways [10, 11]. On the other hand, increased genomic instability has been also reported in LMNA-knockout cells completely lacking lamin A [12].

Here we present a distinct primary progeroid lamino-pathy with overlapping features of HGPS, MAD and RD that is pathophysiologically not related to the accumulation of abnormally processed lamin A species, but rather is characterized by a loss of lamin A resulting in accumulation of dsb and impaired DNA repair.

Results

Discussion

Progeroid phenotypes resembling Restrictive Dermopathy (RD) have been linked to primary or, more frequently, to secondary laminopathies [15]. In primary age-related laminopathies with dominant inheritance, accumulation of mutated prelamin A precursors triggers toxic nuclear effects in a dominant-negative manner resulting in premature aging, more similar to HGPS. In the case of homozygous ZMPSTE24 null mutations, which cause a complete loss of mature lamin A protein, the clinical course of the disease is fatal, foudroyant, and associated with prenatal or neonatal death. Individuals harbouring heterozygous ZMPSTE24 mutations are clinically inconspicuous, as the amount of normal ZMPSTE24 is adequate to process prelamin A to mature lamin A sufficiently [7, 9]. Intriguingly, a case of atypical progeria associated with heterozygous compound mutations in ZMPSTE24 has been described that showed mature lamin A decrease [8].

In the present study, we identified a fatal RD-like disease as a distinct primary progeroid laminopathy linked to the homozygous pathogenic LMNA mutation c.1303C>T (p.R435C). Apparently the heterozygous mutation occurs at a low frequency in the normal population. So far the heterozygous mutation LMNA c.1303C>T (p.R435C) has been associated in one reported case to dilated cardiomyopathy [14]. We therefore performed cardiologic assessment of four of the five heterozygous family members related to the fatality. Except for one individual with mild signs of hypertensive cardiac disease (most likely age related), none of the individuals analysed showed any clinical or echocardiographic evidence of functional or structural cardiac impairment. Additionally, in another study including three heterozygous individuals, no cardiac abnormalities have been reported [13]. Thus, it seems unlikely that the heterozygous LMNA p.R435C mutation alone can be associated to a pathogenic effect. Perhaps additional risk factors are necessary to trigger a cardiomyopathy.

Considering the low frequency of LMNA p.R435C in the normal population, the occurrence of homozygous individuals is extremely rare; assuming Hardy-Weinberg equilibrium, the likelihood was 5×10⁻⁸. However, homozygosity for very rare alleles often occurs on a consanguineous background. This has indeed been found in a Polish family, where the only homozygous LMNA p.R435C has been reported so far [13]. Additionally, as shown for the first time by this study, partial uniparental disomy can result in rare homozygous LMNA mutations.

In contrast to the heterozygous LMNA p.R435C mutation, which expresses a rather mild clinical picture if any, the patient harboring the homozygous mutation presents clinically with a severe progeroid syndrome including severe skin abnormalities as pathognomonic symptoms. Follow-up of the clinical course of the patient for 11 months showed that the phenotype represents a distinct primary progeroid laminopathy, harboring clinical and morphologic overlapping features of RD, HGPS and MAD. The fact that the patient was nearly inconspicuous at birth resembles HGPS; absence of hairiness is typical for RD and HGPS. Skin changes and loss of claviculae have been reported in all three syndromes mentioned, but particularly the generalized tight and thick skin with epidermal thickening, reduction of the rete ridges and rarefication of skin appendages, combined with missing hairiness are pathognomonic features of RD. In contrast to classic RD, the patient had a thickened dermis and subcutaneous lipodystrophy. However, the predominant phenotype of the patient was conditioned by a dramatic progression of the skin abnormalities resulting in microstomia, flexion contractures of the extremities, rocker bottom feet, combined with facial abnormalities like hypolastic nose and micrognathia, all in all resembling RD. Whereas the typical RD patient is diagnosed neonatally in a pre-final stage of disease, similarly the patient developed a “dermal exoskeleton” causing disturbed temperature regulation and global respiratory insufficiency leading to death. In summary, the clinical course of the reported patient appears to one as an accelerated HGPS finally ending up in a delayed RD with overlapping clinical features of MAD.

The patient's progressive skin changes were dramatic and reflected the beginning of a vicious cycle leading to encasement of the patient, loss of mobility, severe growth and developmental delay, muscular hypotension, respiratory impairment and finally death from pneumonia, which is common in RD patients. X-ray analyses at two time points indicated that a continuous degeneration of initially normal appearing bone tissue (claviculae, humeri) occurred. Thus, deformation and absence of bone tissue as a leading symptom of progeroid phenotypes may not be a developmental or differentiation error, but may rather illustrate a symptom of disease progression as it occurs at an advanced stage of the disease.

There are ongoing discussions about the molecular mechanisms of premature aging syndromes including laminopathies and which aspects might resemble physiological human aging. Increased DNA damage, defects in DNA repair mechanisms and shortening of telomeres are characteristics of normal aging. Highlighting one link to the family of laminopathies, telomeres may directly be damaged by Progerin in HGPS (although involvement of telomeres has not been investigated in the present study). Likewise persistent DNA damage is one attribute of other premature aging diseases, even if not all LMNA mutations result in DNA damage and genome instability [16]. However, experimental downregulation of LMNA can lead to accelerated nuclear senescence and apoptosis [17], and altered lamin A function has even been described as an indicator of senescence during physiological aging process [18].

Immuno-histological analyses of tissue samples taken at the beginning and the end of the disease course allowed for the first time a follow-up of functional changes associated with this homozygous LMNA mutation, and thus provided insight into molecular pathogenic processes of progeroid primary laminopathies. We observed a progressive decrease in lamin A levels in the skin of our patient during the worsening of the clinical phenotype (Fig.5). The progression resembles that found in homozygous lamin A knockout mice [19], which were indistinguishable from heterozygous or wild type at birth, but then showed severely retarded postnatal growth and markedly reduced life span. In contrast, the heterozygotes developed apparently normally and did not exhibit any premature mortality when compared to wild type mice. Interestingly, cells from the only LMNA-null patient so far reported [20, 21]showed mislocalization of pRb and early cell cycle arrest possibly due to interference of a cell cycle checkpoint [22].

Activated DNA damage response (DDR) has been discussed as another indicator of replicative senescence and accelerated aging [23]. DDR is thought to play a role in the repair of short-range DNA dsb by binding to the breaks, inhibiting end-resection and facilitating the recruitment of the non-homologous end-joining DNA repair machinery.

It was intriguing that in parallel with the progressive loss of lamin A high levels of DNA dsb occurred in our patient, indicative of increasing DNA damage (Fig.5). It has recently been shown that among other downstream targets p53 signaling pathway can be altered by LMNA dysfunction [24]. Moreover, lamin A is involved in the stabilization of P53 binding protein 1 (53BP1), a component of the DDR pathway [12].

A recent report suggested that the loss of 53BP1 could be responsible for the DNA repair deficiencies observed in LMNA-K.O. cells [25]. Additionally, depletion of lamin A in cultured primary cells has been shown to trigger a senescent cell cycle arrest [26]. Since we found (Fig.6) that following oxidative stress-induced DNA damage very few 53BP1 foci occurred in LMNA R435C transfected cells, it is possible that the DNA repair is delayed or impaired due to the lamin A mutation. It should be pointed out that our results could be in part hampered by an overexpression effect. In fact, a synergistic effect derived from the LMNA mutation combined with protein deficiency appears likely in vivo. For instance, we might expect a reduced importation of 53BP1 into nuclei, which was not observed in LMNA p.R435C transfected fibroblasts. One could speculate that because the aberrant, non-functional, mutant lamin A has a functionally negative effect and likely abnormal conformation, it therefore could also consequently undergo greater degradation than wt-lamin A. The phosphorylation of the histone H2AX at Serine 139 (γH2AX) is the earliest chromatin modification after exposure to ionizing radiation [27] and is thought to be involved in the stable accumulation of 53BP1. But 53BP1 can still be recruited to dsb sites in H2AX null cells, which show only mild defects in DNA damage checkpoint control and DNA repair [28]. Therefore it seems likely that the DNA damage in our patient occurs due to a mis-localization of 53BP1. Unfortunately, we could not use living cells from the patient to test such hypotheses. However, we failed to observe any effect of R435C lamin A in transfected HEK293 embryonic cells, which express very low levels of endogenous lamin A (not shown). This finding argues in favor of a specific role of lamin A in recruiting 53BP1 at DNA damage sites in post-natal cells.

Involvement of this LMNA mutation in DNA damage may be of clinical importance for future patients, as it has been observed that DDR may activate mTOR signaling and in turn drive cellular senescence. Suppression of cellular senescence by using the mTOR-inhibitor Rapamycin has been discussed as an innovative therapeutic approach in the clinical treatment of laminopathies (reviewed in [23]). However, further investigations are needed to assess a possible clinical benefit of patients harboring the reported or other LMNA mutations accompanied by increased DNA damage.

Our data support the idea that the loss-of-Lamin A itself and/or its function in LMNA p.R435C homozygous individuals leads to a loss of DNA-damage-prevention, accompanied by accumulation of double strand breaks, being reflected by progressive degeneration of tissues, osteolysis and cutaneous rigidity as seen in the presented patient. In conclusion, our clinical and functional findings provide a link between the functional loss of lamin A via disturbed DNA repair pathways and degeneration of cells that results in a progeroid phenotype as observed in the presented case. Previously, increased genomic instability has been observed in other progeroid laminopathies including HGPS as well as RD, and is thought to be associated with prelamin A or progerin accumulation [10, 11]. In contrast, our case provides evidence that increased DNA damage/genomic instability in progeroid laminopathies is due to the lack of mature, functional lamin A rather than the abnormal accumulation of prelamin A forms.

Materials and Methods

Supplementary Materials

Supplemental File