Background
The studies of clinical and cellular consequences of
LMNA mutations in humans have provided several indications of a close physiopathological relationship between premature aging and lipodystrophic syndromes. Indeed, human naturally-occuring
LMNA mutations, among other phenotypes of laminopathies, have been shown to be responsible for premature ageing syndromes (Hutchinson-Gilford progeria, HGPS, and mandibuloacral dysplasia, MAD) [
1‐
4], lipodystrophic syndromes (familial partial lipodystrophy of the Dunnigan type, FPLD2) [
5,
6], and mixed phenotypes [
7‐
10]. At the cellular level, a highly similar disorganization of the nuclear lamina is observed in fibroblasts from patients with FPLD2, MAD and HGPS [
3,
11,
12], including cellular replicative senescence and prelamin A accumulation [
13‐
15]. Although the pathophysiological mechanisms leading to
LMNA-linked premature aging are not fully understood, a major hypothesis is that alterations in maturation and farnesylation of lamin A induce genomic instability, abnormal epigenetic control of heterochromatin and DNA damage responses, and mesenchymal stem cell defects [
16‐
18].
Werner syndrome (WS) is an autosomal recessive premature aging syndrome due to biallelic inactivating mutations in
WRN, encoding a RecQ DNA helicase/exonuclease involved in DNA replication and repair [
19]. Its prominent features, occurring after adolescence, associate “bird-like” facies, scleroderma-like skin changes with tight and atrophic skin, bilateral cataracts, short stature and premature greying of scalp hair [
20]. An initial clinical presentation as a lipodystrophic syndrome has not been previously described.
In women with familial partial lipodystrophies, decreased fertility and obstetrical complications have been reported to be mainly linked to insulin resistance and metabolic disturbances, with an increased prevalence of polycystic ovary syndrome, gestational diabetes and eclampsia [
21,
22]. The mechanisms leading to decreased fertility in Werner syndrome have not been deciphered. Only rare cases of pregnancies have been reported in women with probable, but not genetically-confirmed Werner syndrome [
23‐
26].
Here we report the cases of two women investigated for lipodystrophy and severe insulin resistance, which revealed Werner syndrome due to homozygous or compound heterozygous, non-sense or frameshift mutations in the WRN gene. The two patients became pregnant after initiation of metformin therapy. Both pregnancies were complicated by cervical incompetence, leading to a second-trimester abortion in one case and to a preterm delivery in the other patient. Cultured fibroblasts obtained from one patient showed cellular senescence, nuclear dysmorphies, and lamin staining abnormalities similar to those found in laminopathies, but did not overexpress immature prelamin A.
Our study points out that primary defects in DNA replication and/or repair should be considered as possible causes of lipodystrophic syndromes with extreme insulin resistance. In addition, we show that cell nuclear dysmorphies with alterations in lamin staining can be secondary to cellular senescence of different origin.
Discussion
In the present study, we add further evidence for a pathophysiological link between cellular senescence and lipodystrophy, by showing for the first time that partial lipodystrophy syndrome with extreme insulin resistance can be the initial referring presentation of the adult progeria Werner syndrome, due to a primary defect in the WRN enzyme, involved in DNA replication and repair.
Lipodystrophic syndromes are heterogeneous diseases of genetic or acquired origin, characterized by generalized or partial lipoatrophy associated with insulin resistance. Recent advances in molecular genetics have shown that primary defects in fat differentiation and/or adipose lipid droplet formation or maintenance are the main causes of genetic lipodystrophies (for review, see [
32‐
34]). However, the hypothesis that lipodystrophy could also be secondary to primary mesenchymal cellular senescence was raised by the studies of laminopathies, which collectively name a group of diseases due to alterations in the ubiquitous nuclear intermediate filaments A type-lamins, encoded by the
LMNA gene. Indeed, mutations in
LMNA can lead to Dunnigan-type familial partial lipodystrophy (FPLD2) [
5,
6], but also to accelerated ageing syndromes [
1‐
4], and to mixed overlapping phenotypes with both lipoatrophy, metabolic complications and progeroid signs [
7‐
10]. In addition, cellular studies have shown that, although lamin A alterations could impair adipogenesis through mislocalisation of the key adipogenic transcription factor SREBP1c [
14,
35‐
37], premature cellular senescence probably also participates to the pathophysiology of
LMNA-linked lipodystrophy [
15].
Werner syndrome, also called adult progeria (OMIM 277700), is one of several progeroid syndrome due to defective DNA helicases (for review, see [
20]). This segmental aging syndrome, affecting several organ systems, is due to recessive null mutations in the WRN protein, which exhibits exonuclease, ATPase and helicase activities. Cellular senescence associated with Werner syndrome has been linked to DNA replication and repair defects [
19]. The clinical diagnostic criteria have been defined by the International Registry of Werner Syndrome (
http://www.wernersyndrome.org/registry/diagnostic.html) [
20], and have been recently revised on the basis of the results of a Japanese nationwide epidemiological survey [
38].
Both patients described here were referred for partial lipodystrophy, which is not listed as a classical sign of the disease. Several lipodystrophic features were different to those usually observed in other types of partial lipoatrophic syndromes, as FPLD2 or 3 due to
LMNA or
PPARG mutations, respectively. Indeed, in both patients, peripheral lipoatrophy was associated with loss of limb muscles, which contrasted with the muscle hypertrophy associated with FPLD2 and 3. In addition, marked central adiposity was striking, and imagery revealed an asymmetrical distribution of subcutaneous fat in the thighs. Both patients also exhibited several cardinal signs of Werner disease,
i.e. bilateral cataracts, tight and atrophied skin with hyperkeratosis, and premature greying of scalp hair. In addition, patient 2 had short stature, and two siblings of patient 1 were probably affected, although molecular analysis was not possible. Further signs, listed as reminiscent of Werner disease, were also present: atherosclerosis and altered fertility in both cases, and high-pitched voice, diabetes, osteoporosis and mesenchymal neoplasm in patient 2. In both patients, we identified truncating null mutations affecting both alleles of
WRN gene with loss of the nuclear localization signal. The homozygous p.Q748X
WRN mutation of patient 1 was previously found in a Caucasian man diagnosed with Werner syndrome, but his clinical features were not reported [
20], whereas patient 2 was affected by new
WRN mutations. No evident genotype-phenotype correlations have been reported in Werner syndrome [
20,
39], although proximal truncation of WRN protein could lead to severe phenotypes [
40]. Further studies are needed to eventually link the lipodystrophic clinical presentation to specific
WRN mutations. However, our report shows that Werner syndrome is an important differential diagnosis in patients initially presenting with partial lipodystrophy as a prominent feature, leading to a specific follow-up, in particular regarding cancer risk, gynecology, and genetic counseling.
These two women presented with severe insulin resistance, with insulinemia being dramatically increased during OGTT without hypoglycemia. Low SHBG and adiponectin levels suggest a post-receptor insulin signalling defect, as observed in other lipodystrophic syndromes, where ectopic fat deposition, particularly in muscle and liver, is thought to play a major role in insulin resistance [
41]. In accordance, our patients had also liver steatosis and hypertriglyceridemia. Their peripheral skinfolds confirmed a severe subcutaneous lipoatrophy of the limbs, whereas percentage of fat measured by DEXA in limbs was increased or only slightly decreased, suggesting that the limb muscles could be infiltrated with lipids. Insulin resistance has been previously reported in patients with Werner syndrome, with hypoadiponectinemia and increased intra-abdominal visceral fat in some patients [
42,
43], but insulin values did not reach such dramatically elevated levels [
43,
44]. Therefore, our report suggests that the presence of a marked lipodystrophy can contribute to the severity of insulin resistance in Werner syndrome.
Our report also points out the specific gynecological and obstetrical complications of Werner syndrome. Hypogonadism is a classic sign of Werner syndrome, but its precise origin has not been investigated [
20,
38]. To our knowledge, only three pregnant women with clinically-suspected, but not genetically-confirmed Werner syndrome, have been previously reported [
23‐
26]. None of them carried their pregnancies to full-term, with spontaneous abortions at 10, 16 and 23 weeks [
23], or preterm delivery at 25 to 34 weeks of gestation, due to cervical insufficiency in three cases [
23,
24] or to caesarean section for maternal life-threatening ischemic heart disease in one case [
25,
26]. Preeclampsia occurred during two pregnancies, in the absence of diabetes [
23], and placental vascular alterations were observed in one case [
24]. Our two patients had experienced early spontaneous abortions and had reduced fertility. They both became pregnant during the follow-up, but severe cervical incompetence, which thus appears to be a frequent obstetrical complication in this disease, led to a preterm birth in one case, and to a second term-abortion in the other. No specific abnormality was evidenced in the foetus and the preterm newborn. Pancreatic islet hyperplasia observed at foetal autopsy was linked to gestational diabetes. The placenta showed signs of chorioamnionitis and funiculitis, which were secondary to premature rupture of membranes.
In these women, reduced fertility was probably related to a premature decrease in the pool of primordial ovarian follicles (diminished ovarian reserve). Indeed, although their menstrual cycles were not modified, and their FSH levels were still within normal range, indicating that they did not had premature ovarian failure, their AMH and inhibin B serum levels were very low [
45]. Biological ovarian hyperandrogenism, observed in patient 2, and polycystic ovaries in patient 1, favored by extreme insulin resistance, could also have contributed to decreased fertility. In accordance, both pregnancies were obtained after reducing hyperinsulinemia with metformin treatment. Therefore, both insulin resistance and premature ovarian ageing could lead to decreased fertility in Werner syndrome.
Lipodystrophic and progeroid laminopathies are characterized by cellular senescence and nuclear dysmorphies, with nuclear blebs showing abnormal nuclear staining of A and B-type lamins [
3,
11,
12,
15,
46]. Several studies have suggested that accumulation of farnesylated forms of prelamin A could underlie these abnormalities (for review, see [
47]). Our present results show that fibroblasts with WRN null mutations present nuclear deformations similar to those observed in laminopathies, as described in one previous study [
48]. We also showed that oxidative stress and cellular senescence were enhanced in
WRN-mutated cells. However, prelamin A was not accumulated in
WRN-mutated cells, suggesting that primary DNA repair defects and
LMNA alterations act by different mechanisms to induce premature ageing. In addition, although lamin B staining was decreased in nuclear blebs, the overall lamin B1 protein expression was increased in
WRN-mutated cells, which was not previously described in laminopathies. The role of B type-lamins in cellular senescence is complex [
49]. Indeed, replicative or oncogene-induced senescence has been linked to underexpression of lamin B1 [
50], but oxidative stress-induced senescence to overexpression of lamin B1 [
51]. An interesting hypothesis is that cellular stresses which alter the normal ratio of lamin B1 to functional lamin A, lead to deleterious changes in nuclear lamina, then senescence [
49]. In accordance, and in line with our results, cells from patients with ataxia-telangiectasia, another genetic disease due to DNA damage signalling defects, display a state of endogenous oxidative stress which induces lamin B1 overexpression, nuclear shape alterations and senescence through MAP kinase activation [
51]. It is possible that MAP kinase activation, which was previously observed in Werner syndrome [
52], could also contribute to lamin B1-linked cellular senescence. Whether
WRN-linked lipodystrophy could be secondary to primary mesenchymal cellular senescence, as it was suggested in laminopathies, needs to be further investigated.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
BD cared for one of the patients, analyzed the data, contributed to the discussion and drafted the manuscript. PD’A, MS, SO, GC, PB, PL, BC and SC-M cared for one of the patients, contributed to the discussion and reviewed the manuscript. MA carried out the cellular studies and contributed to the discussion, NU performed molecular analyses, contributed to the discussion and reviewed the manuscript. Y-JB performed molecular analyses, cared of one of the patient, contributed to the discussion and reviewed the manuscript. RG performed pathological analyses, contributed to the discussion and reviewed the manuscript. CV cared of the patients, analyzed the data, and wrote the manuscript. All authors read and approved the final manuscript.