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Department of Endocrinology and Metabolism (M.C.V., J.L.W.); Laboratory of Endocrinology (P.P.); Departments of Neurology (T.S., P.V.), Cardiology (A.M.), and Pediatrics (J.M.C.); and Laboratory of Neurological Pathology (C.A.M., N.R.-E.), Lille University Hospital, 59037 Lille, France; and Institut National de la Santé et de la Recherche Médicale, Unité 402, Saint Antoine Faculty of Medicine (O.L., J.C., C.V.), and Molecular Biology Laboratory, Saint Antoine Hospital (O.L.), Pierre and Marie Curie University, 75012 Paris, France
Address all correspondence and requests for reprints to: Dr. M. C. Vantyghem, Department of Endocrinology and Metabolism, Lille University Hospital, 6 rue du Prof. Laguesse, 59037 Lille Cedex, France. E-mail: mc-vantyghem{at}chru-lille.fr.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Lamin A/C mutations induce nuclear disorders affecting the lamina and the relationships with its partners, such as chromatin, in both humans and mice (17, 18, 19, 20, 24, 25, 26, 27). However, these cellular alterations are not specific for the different clinical phenotypes of laminopathies.
Three-dimensional structural studies of the lamin A/C carboxyl-terminal end, featuring a conformation similar to that of an immunoglobulin fold, showed that FPLD-associated mutations cluster on the external surface of the molecule, whereas most muscular disease-associated mutations lie within the domain and predictably cause major structural perturbations (28, 29). An interesting hypothesis is that muscular laminopathies could result from a lamin A/C loss of function, whereas FPLD could be due to altered interactions between mutated lamins and an adipose-specific partner (30, 31). Alternatively, a possibly disturbed interaction between Myne 1, a muscle-specific inner nuclear membrane protein, and mutated lamins could cause the neuromuscular abnormalities (32).
Neuromuscular and cardiac phenotypes of FPLD patients carrying the most frequent mutations at codon 482 of LMNA have not yet been systematically studied. Awareness of potential risks is a prerequisite for correct clinical management of patients, especially at the cardiac level. Muscular or cardiac involvement in typical FPLD also raises questions about the pathophysiology of the disease.
We present here the phenotypes, in particular at the neuromuscular and cardiac levels, of 14 subjects from two unrelated families, 10 of them bearing the FPLD-linked LMNA R482W heterozygous mutation.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Two probands from two unrelated families, H and K (HIII-1 and KIII-1), were previously referred to our department for lipodystrophy and/or diabetes and were diagnosed with FPLD due to the R482W heterozygous mutation of the LMNA gene (10). We studied here 14 relatives from these families (Fig. 1
). Eight adults (HII-2, HII-3, HII-5, HIII-1, KII-2, KII-3, KIII-1, and KIII-3) and two children (HIII-5 and HIII-7) carried the LMNA mutation, whereas three adults (KII-1, KIII-2, and KIII-4) and one child (HIII-6) were unaffected. All individuals or their parents gave their informed consent for the study.
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Subjects underwent a complete clinical examination, including anthropometric measurements and evaluation of Ferriman-Gallwey score. Metabolic investigations were performed after a 12-h fasting period and included plasma measurements of glucose, insulin at 0 and 120 min of a 75-g oral glucose tolerance test, lipid profile (total cholesterol and triglycerides), and leptin. Serum insulin concentrations were measured using a highly specific immunoradiometric assay (Bi Insulin IRMA, Cis Bio International, Gif sur Yvette, France) with a proinsulin cross-reactivity less than 0.005%. Serum leptin levels were measured using an RIA (Sensitive Human Leptin RIA kit, Linco Research, Inc., St. Charles, MO).
Genomic DNA from each individual was isolated from peripheral blood lymphocytes using a commercial kit (QIAamp DNA Blood MaxiKit, Qiagen, Courtaboeuf, France) according to the manufacturer’s protocol. It was screened for the LMNA R482W mutation using direct sequencing and/or the PCR-restriction fragment length polymorphism method using the MspI enzyme, as previously described (10). The entire LMNA coding sequence was sequenced in several patients, including HII-3.
Neuromuscular investigations
Specialized neuromuscular examination and blood measurement of creatine phosphokinase were performed in each individual, and electromyogram of the four limbs was performed in each LMNA-mutated adult. Muscular magnetic resonance imaging (MRI) was performed in patients KII-3, KIII-1 (calves), and HII-2 (thighs). Neurological examination was performed by a neurologist who did not know the genotype of the subjects at the time of examination.
Muscle biopsies were performed in peroneal (patient HII-5) or deltoid (LMNA-mutated patients HII-2, HII-3, HIII-1, KII-3, and KIII-1, and non-LMNA-mutated relatives KII-1, KIII-2, and KIII-4) muscles. Muscle samples were snap-frozen by immersion in liquid nitrogen-cooled isopentane and were kept at –80 C until used. Quadriceps muscle samples from healthy age-matched subjects were used as controls. All patients had authorized the use of muscle samples for research.
Histochemistry stains were performed on serial 10-µm thick transverse cryostat sections.
Immunohistochemistry was performed on 5-µm thick sections, using a streptavidin-biotin complex technique coupled to horseradish peroxidase in a Ventana ES200 automat (Ventana Medical Systems, Tucson, AZ). All transverse frozen sections were immunolabeled on the same slide. Antibodies directed against dystrophin; glycoprotein associated with dystrophin; sarcoglycan-
, -ß, and -
; dysferlin; emerin; and lamin A/C were purchased from Novocastra (Newcastle, UK). Negative control labeling was achieved using incubation with nonimmune serum.
Western blotting was carried out using a denaturing 4–7% SDS-PAGE. After electrophoresis, the gels were blotted, and the membrane was probed with primary antibodies directed against calpain 3 (NCL-CALP-2C4) or emerin (both purchased from Novocastra), then with a peroxidase-conjugated secondary antibody (goat antimouse immunoglobulin G H+L, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Immunoreactive bands were visualized using the enhanced chemiluminescence detection system (Amersham Biosciences, Little Chalfont, UK).
Cardiac investigations
Patient evaluation included clinical examination, electrocardiogram, echocardiography (M-mode, two-dimensional, and Doppler), and 24-h blood pressure and rhythm monitoring.
Calpain 3 gene study
All 24 exons and surrounding intronic sequences, and the proximal promoter from the calpain 3 gene (CAPN3) were amplified, then directed sequenced in genomic DNA from patient HII-3. PCR was carried out using intronic primer pairs as previously described by Richard et al. (33). An additional specific primer pair (prom forward, 5'-agtgcagagattagagacgtg-3'; prom reverse, 5'-gaaagagagtgagggttgtgt-3') was designed to amplify the –605/–11 fragment of the CAPN3 proximal promoter that contains the main cis-regulatory elements (33). The amplicons were first visualized in an ethidium bromide-stained agarose gel before being purified on microcolumns (Microcons YM-50, Millipore Corp., Bedford, MA) according to the manufacturer’s instructions. Both strands of the purified amplicons were sequenced using the ABI PRISM model 377XL automatic sequencer and the ABI PRISM AmpliTaq FS Big Dye Terminator cycle sequencing ready reactions kit (Applied Biosystems, Courtaboeuf, France).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The clinical phenotype of partial lipodystrophy, characterized by a particular habitus with peripheral sc lipoatrophy contrasting with accumulation of fat in the face and neck, was only present in patients bearing an LMNA R482W mutation. All LMNA-mutated women were lipodystrophic, whereas LMNA-mutated men showed preservation of some peripheral sc fat, although this was slightly reduced in HII-2. However, a progressive neck enlargement from puberty was reported in both male and female patients. All LMNA-mutated women, but none of the unaffected women, presented with noticeable broad shoulders and short legs, assessed by the values of biacromial/bitrochanterian diameter (>1) and total height/leg length ratio (>2), respectively. Thick hands with broad, infiltrated, spindle-shaped fingers (Fig. 2A) were also only found in FPLD women. The body fat repartition was clinically normal in all prepubertal children.
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Leptinemia was above the normal range in the two obese individuals, either LMNA-mutated (HII-2) or not (KII-1). Nonobese patients showed normal or low leptin levels. Interestingly, leptin secretion was relatively preserved in patient HII-3, who was cachectic [body mass index (BMI), 12 kg/m2], but presented a facio-cervical distribution of fat and severe myopathy, as described below (Fig. 2B
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In accordance with previous reports, subjects with the LMNA R482W mutation showed a high prevalence of insulin resistance, diabetes, and hypertriglyceridemia. However, these metabolic disturbances are also frequent in the general population; subject KII-1 presented with obesity, severely complicated, insulin-treated type 2 diabetes, and combined hyperlipidemia but was exempt of FPLD. LMNA-mutated adults had a BMI less than 25 kg/m2 (except in one patient), but oral glucose tolerance test results were normal only in two patients (KII-3 and KIII-3). The other six patients showed hyperinsulinemia (HIII-1, HII-2, and HII-5) and/or glucose intolerance (HII-2 and HII-5) or diabetes (HII-3, KII-2, and KIII-1). The control of hyperglycemia was achieved with diet alone in one case (HII-3), but was very difficult to obtain despite large insulin doses in another case (KIII-1) (34). The severity of hypertriglyceridemia (and hirsutism in women) mostly paralleled the degree of glucose metabolism impairment, with a normal range of triglyceride levels in patient KII-3, mild to moderate hypertriglyceridemia in six FPLD patients, and major hypertriglyceridemia with an antecedent of acute pancreatitis in patient KIII-1. The metabolic results of the two prepubertal boys carrying the LMNA R482W mutation (HIII-5 and HIII-7) were not different from those of their unaffected brother (HIII-6).
Neuromuscular investigations (Table 2)
Medical history and clinical examination.
Two patients (HII-3 and HII-4) were diagnosed at age 8 yr with myopathy. Patient HII-3 had been suffering from LGMD, with initial calf hypertrophy, proximal limbs weakness, and both shoulder and pelvic girdle amyotrophy. Progressive worsening of girdle amyotrophy led to loss of ambulation at age 16 yr, then major functional disability with respiratory insufficiency requiring nocturnal oxygenotherapy (Fig. 2B). No precise neurological data or LMNA genotype were available for her sister (HII-4), who died at age 33 yr with severe myopathic symptoms.
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), and rolling gait related to lower limb proximal weakness (Trendelenburg’s sign) was found in six patients, all of them bearing the LMNA R482W mutation. Clinical peripheral distal neuropathy was confirmed in patients KII-1 and KIII-1, who were known to have multicomplicated diabetes. The neurological examination was normal in relatives who did not carry a mutation in LMNA as well as in the prepubertal LMNA-mutated boys HIII-5 and HIII-7.
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Electromyogram. Myogenic disturbances were observed in patient HII-3 (with severe LGMD), whereas electrical signs of distal peripheral neuropathy were found in diabetic patients KII-1 and KIII-1, in accordance with the clinical findings.
Muscle MRI.
Muscle MRI was performed in three LMNA-mutated adults. In patient HII-2, MRI of the thighs showed major atrophy of the left adductor magnus and moderate muscular fatty degeneration of the back part of the thighs, a sign previously described in LGMD (35). At the calf level, MRI revealed muscular hypertrophy with a slight muscular fatty infiltration and a near-total disappearance of sc adipose tissue in patient KII-3. MRI was considered normal in patient KIII-1, apart from lack of sc adipose tissue (Fig. 3B).
Muscle biopsies.
A muscle biopsy was performed in six LMNA-mutated and three non-LMNA-mutated adults (Fig. 4). For LMNA-mutated patients, we observed a dystrophic aspect of the muscle, with variations in fiber size, major interstitial fibrosis (75%), and cellular necrosis and regeneration in patient HII-3 (Fig. 4, A and B). The protein expression and cellular localization of dystrophin, the antiglycoprotein associated with dystrophin, dysferlin, emerin, and lamin A/C, as assessed by immunostaining, were not different in patient HII-3 and control subjects (Fig. 4C). However, the muscular protein expression of calpain 3 was not detected by Western blot in patient HII-3 as opposed to controls, whereas emerin was expressed at a normal level (Fig. 5). In patient HII-5, muscle histological studies showed an excess of oxyphilic granules (revealed by Gomori staining) in type I fibers, suggesting an increased amount of mitochondria. Hypertrophy of type IIA and IIB fibers contrasting with a grouping of hypotrophic type I fibers was also observed. An excess of lipid droplets was also noticed between type II myofibrillas. Less than 1% of fibers were atrophic, rugged, degenerated, or necrotic. The results were compatible with muscular dystrophy (Fig. 4B). No immunohistochemical or Western blot studies could be performed because of the small size of the sample. The biopsies of the other four LMNA-mutated patients only revealed an increase in the contents in lipid droplets without vacuolization in two of them (Table 2 and Fig. 4, A and B). The expression and nuclear localization of emerin and lamin A/C as well as the protein amount of calpain 3 were not different from those in controls (Fig. 4C).
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Taken as a whole, these neuromuscular studies showed the following. 1) Patient HII-3, bearing the LMNA R482W mutation, presents a typical FPLD associated with clinical and histological findings allowing the diagnosis of severe progressive LGMD. Muscular calpain 3 expression was not detectable. 2) Patient HII-5, bearing the LMNA R482W mutation, presents a typical FPLD associated with clinical and histological findings compatible with a moderate form of progressive LGMD. 3) In addition to these two patients, five of the six other LMNA-mutated adult patients exhibited clinical and biological signs of FPLD and neuromuscular findings compatible with mild forms of progressive LGMD, as suggested by the clinical association of calf hypertrophy, perihumeral muscular atrophy, and lower limb proximal weakness responsible for a rolling gait, which was not found in any of the subjects exempt from the LMNA mutation. In one of these patient (HII-2), MRI of the thighs showed muscular atrophy of gluteus maximus and thigh adductors associated with fatty degeneration (also present in patient KII-3; Fig. 3B), which also suggests the diagnosis of LGMD.
Interestingly, the severity of the lipodystrophy and the muscular phenotype were not related. However, the presence of lipid droplets in muscle, a nonspecific finding, could be related to the state of insulin resistance, because we found a similar aspect in subject KII-1, with common type 2 diabetes and obesity, whereas two non-LMNA-mutated, nondiabetic subjects had normal muscle histology.
Cardiac investigations (Table 3)
Several patients had an abnormal cardiac examination, but most of the symptoms were of low specificity. Cardiac septal hypertrophy was found in three patients with FPLD, whereas a more common form of concentric left ventricular hypertrophy was seen in patient KII-3, who had high blood pressure. Several patients presented with premature atherosclerosis: an ischemic cerebral stroke had occurred in the FPLD patients HII-2 and KII-3 in their forties, and non-FPLD patient KII-1 had coronaropathy. However, patient KIII-3, 24 yr old, was affected with a symptomatic second degree atrioventricular block that could be related to a cardiac consequence of his laminopathy.
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No mutation was found in the whole coding sequence, in the intron-exon boundaries, or in the proximal promoter region (–605/–11) of CAPN3 in patient HII-3.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The aim of our study was to determine the specific characteristics of the phenotype, particularly at the muscular and cardiac levels, of patients bearing the FPLD-linked heterozygous LMNA R482W mutation. We studied 14 subjects belonging to two unrelated families, 10 of them (eight adults and two children) exhibiting this LMNA mutation.
Partial lipodystrophy, with sc lipoatrophy of limbs and trunk and fat accumulation in the cervico-facial area, was a major phenotypic trait in females with the FPLD-linked LMNA mutation, but was less striking in males, as previously reported (10, 36). However, a progressive neck enlargement from puberty was constant in either gender in our patients. In addition, thick hands with broad, spindle-shaped fingers are frequently seen in FPLD females, a fact that has not been pointed out in previous reports. Metabolic disorders were also generally more severe in FPLD females than in males, although nonspecific and highly variable; among two LMNA-mutated women, one (KII-3) had normal insulinemia and glucose tolerance at age 49 yr, whereas her young and lean niece (KIII-1; 19 yr old; BMI, 19 kg/m2) was affected by a severe, multicomplicated, insulin-resistant diabetes. The severity of hypertriglyceridemia and hyperandrogenism in FPLD females was roughly correlated with the severity of insulin resistance. However, hypertriglyceridemia could be found before the deterioration of glucose tolerance, as previously reported (36, 37). As expected, our FPLD patients, in contrast to generalized lipodystrophic patients, did not have extremely low leptin levels (38). Prepubertal children were asymptomatic, which is consistent with the widely accepted pubertal or postpubertal appearance of the first clinical signs in FPLD.
We found clinical muscular alterations in all eight LMNA R482W-mutated adults, but in none of the control relatives or the children. One patient with FPLD (HII-3) presented from childhood with a very severe LGMD, compatible with the 1B form, characterized by weakness, then amyotrophy of shoulder and pelvic girdles, and calf hypertrophy. However, cardiac conduction defects that are usually, but not always, associated with LGMD 1B, were lacking in this patient. The preferential proximal limb-girdle alterations of the musculature and the absence of contractures argue against EDMD (13, 14). Histological findings were not in favor of a spinal muscular atrophy, and the normal immunostaining for dystrophin and glycoprotein associated with dystrophin excluded dystrophinopathies and sarcoglycanopathies (LGMD 2D to 2F).
Unexpectedly, in this patient, we found a defect in the muscular expression of calpain 3, without any alteration in the coding sequence, intron-exon boundaries, or proximal promoter regions of the calpain 3 gene. This suggests that the LMNA mutations could secondarily alter calpain 3 expression, as it was previously shown in myopathies due to mutations in the dysferlin gene, responsible for LGMD 2 (39). A low level of expression of calpain 3 in skeletal muscle was shown to be associated with insulin resistance (40), but in our study it seems more likely to be related to the severity of the myopathy, because patient KIII-1, with milder muscular abnormalities, but more severe insulin resistance, showed a normal expression of muscular calpain 3.
Muscular anomalies of various severity affected FPLD patients, but not control relatives, from the two unrelated families, in favor of an involvement of the LMNA R482W mutation in the muscular phenotype. Accordingly, a postmortem study performed by Haque et al. (41) showed a muscular atrophy of the quadriceps femoris and anterior diaphragm muscles in a 66-yr-old woman with FPLD due to an LMNA R482Q mutation. The clinical association of calf hypertrophy, perihumeral muscular atrophy, and rolling gait related to lower limb proximal weakness was suggestive of mild forms of LGMD in six of our FPLD patients. Among them, two patients spontaneously complained of proximal muscular weakness, and one (KII-3) spontaneously asked to be referred to a neurologist. Muscle histology revealed a nonspecific dystrophic aspect with variations in fiber diameter in one FPLD patient, which might be related to LGMD (42). However, the excess of muscular lipid droplets found in two other LMNA-mutated patients is a nonspecific finding that could be a consequence of muscle weakness and inactivity, but might also result from and contribute to insulin resistance (43). In accordance, muscular lipid droplets were also observed in subject KII-1, who was exempt of FPLD but presented with common obesity and diabetes, but were absent from muscular samples of two non-LMNA-mutated subjects with normal glucose tolerance. The muscular protein expression of lamin A/C and its partner, emerin, was not altered, nor was their nuclear localization, assessed by immunochemistry. Similar results were previously shown in LMNA-linked myopathies using Western blot and conventional fluorescence microscopy, respectively (44, 45).
Cardiac investigations confirmed the previously reported increased risk of premature atherosclerosis in our FPLD patients (11). Other cardiac alterations could be nonspecific, although the presence of a second degree atrioventricular block in a 24-yr-old man with normal glucose tolerance could be related to his lamin A/C alteration.
In conclusion, this study points out that most patients affected by the FPLD-linked R482W LMNA mutation could show, in addition to the lipoatrophic phenotype, a myopathic phenotype of various severity. These phenotypes do not have similar natural history, because myopathy could be present from childhood, whereas FPLD symptoms appear only after puberty. A wide clinical spectrum of muscular features might be encountered, ranging from invalidating myopathy of early disclosure to mild delayed-onset muscular dystrophy. In addition, the severity of lipodystrophy, myopathy, and cardiac signs were not related. Careful neuromuscular and cardiac examination with echocardiography and 24-h electrocardiogram monitoring should be carried out in FPLD patients, even in those bearing the typical LMNA R482W mutation. These descriptions suggest that FPLD could be a not so strict adipose tissue-originating disease and raise new questions concerning the involvement of an exclusively adipocyte-expressed partner of lamin A/C in the pathophysiology of the disease.
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Footnotes |
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Received September 23, 2003.
Accepted July 25, 2004.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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gene in a patient with familial partial lipodystrophy. J Clin Endocrinol Metab 87:408–411. Diabetes 52:910–917-helical rod domains of the lamin A/C gene. Am J Med 112:549–555[CrossRef][Medline]
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