LMNA Mutations in Patients with Severe Metabolic Syndrome

0021-972X/07/$15.00/0 Printed in U.S.A.

The Journal of Clinical Endocrinology & Metabolism 92(12):4835– 4844 Copyright © 2007 by The Endocrine Society doi: 10.1210/jc.2007-0654

New Metabolic Phenotypes in Laminopathies: LMNA Mutations in Patients with Severe Metabolic Syndrome Aure´lie Decaudain, Marie-Christine Vantyghem, Bruno Guerci, Annie-Claude Hećart, Martine Auclair, Yves Reznik, Herve´ Narbonne, Pierre-Henri Ducluzeau, Bruno Donadille, Ce´leste Lebbe´, Ve´ronique Be´re´ziat, Jacqueline Capeau, Olivier Lascols, and Corinne Vigouroux INSERM U680 (A.D., M.A., B.D., V.B., J.C., O.L., C.V.), Paris, F-75012 France; Universite´ Pierre et Marie Curie-Paris6, Faculte de Me´decine, UMR 5680 (A.D., M.A., B.D., V.B., J.C., O.L., C.V.), Paris, F-75012 France; AP-HP, Hoˆpital Tenon, Service de Biochimie et d’Hormonologie (J.C., C.V.), Paris, F-75020 France; Service d’Endocrinologie et Maladies Me´taboliques (M.-C.V.), Centre Hospitalier Universitaire (CHU) de Lille, F-59037 France; Hoˆpital Jeanne d’Arc (B.G.), Service de Diabe´tologie, Endocrinologie et Nutrition, CHU Nancy, F-54201 France; Service d’Endocrinologie, Maladies Me´taboliques et Me´decine Interne (A.-C.H.), CHU de Reims, F-51092 France; Service d’Endocrinologie et Maladies Me´taboliques (Y.R.), CHU Coˆte de Nacre, F-14033 France; Hoˆpital Sainte Marguerite, Assistance Publique-Hoˆpitaux de Marseille, Service de Nutrition, Maladies Me´taboliques, Endocrinologie (H.N.), F-13274 Marseille, France; Centre de Recherche en Nutrition Humaine (P.-H.D.), Hospices Civils, Lyon, F-69002 France; AP-HP, Hoˆpital Saint-Louis (C.L.), Service de Dermatologie, Paris, F-75010 France; and AP-HP, Hoˆpital Saint-Antoine, De´partement de Biologie Molećulaire (O.L.), F-75571 Paris, France Context: Mutations in the LMNA gene are responsible for several laminopathies, including lipodystrophies, with complex genotype/ phenotype relationships. Objective, Design, Setting, and Patients: Sequencing of the LMNA coding regions in 277 unrelated adults investigated for lipodystrophy and/or insulin resistance revealed 17 patients with substitutions at codon 482 observed in typical Dunnigan’s familial partial lipodystrophy and 10 patients with other mutations. We report here the phenotypes of the patients with non-codon 482 mutations and compare them with those of 11 patients with codon 482 mutations. We also studied skin fibroblasts or lymphocytes from seven patients. Results: LMNA mutations found in nine patients studied here affected the three protein domains. Eight of them were novel. The 10 patients with non-codon 482-associated mutations fulfilled the International Diabetes Federation diagnosis criteria for metabolic syn-

L

AMINS A AND C, the two main isoforms of A-type lamins, are produced by alternative splicing of the LMNA gene (1). A-type lamins, expressed in most differentiated cells, polymerize with B-type lamins to form the nuclear lamina, a filamentous network located at the nucleoplasmic side of the inner nuclear membrane, involved in nuclear organization and functions (reviewed in Ref. 2). Mutations in the LMNA gene have been shown to be responsible for several distinct diseases, called laminopathies, belonging to the groups of skeletal and/or cardiac muscular dystroFirst Published Online August 21, 2007 Abbreviations: BMI, Body mass index; CT, computed tomography; DCM-CD, dilated cardiomyopathy with conduction defects; DEXA, dual-energy x-ray absorptiometry; FPLD2, familial partial lipodystrophy of the Dunnigan type; HDL, high-density lipoprotein; NLS, nuclear localization signal domain; OGTT, oral glucose tolerance test. JCEM is published monthly by The Endocrine Society (http://www. endo-society.org), the foremost professional society serving the endocrine community.

drome. Most of them lacked the typical lipoatrophy observed in Dunnigan’s familial partial lipodystrophy. However, the severity of insulin resistance, altered glucose tolerance, and hypertriglyceridemia and the alterations of cell nuclei were similar in patients with codon 482- and non-codon 482-associated mutations. Calf hypertrophy, myalgia, and muscle cramps or weakness were present in nine patients and cardiac conduction disturbances in two patients with non-codon 482 LMNA mutations. Conclusions: We describe here new phenotypes of metabolic laminopathy associated with non-codon 482 LMNA mutations and characterized, in the absence of obvious clinical lipoatrophy, by severe metabolic alterations and frequent muscle signs (muscular hypertrophy, myalgias, or weakness). Dual-energy x-ray absorptiometry and/or cross-sectional abdominal and thigh imaging can help diagnosis by revealing subclinical lipodystrophy. The prevalence and pathophysiology of metabolic laminopathies need to be studied further. (J Clin Endocrinol Metab 92: 4835– 4844, 2007)

phies [Emery-Dreifuss muscular dystrophy (3), limb-girdle muscular dystrophy type 1B (4), and dilated cardiomyopathy with conduction defects (DCM-CD) (5)], axonal neuropathies [Charcot-Marie-Tooth disease type 2 (6)], familial lipodystrophies (7, 8), and premature aging syndromes [mandibuloacral dysplasia (9), Hutchinson-Gilford progeria (10, 11), and restrictive dermopathy (12)]. The precise pathophysiology of laminopathies remains elusive, but increasing evidence shows that it involves alterations in cellular mechanical stress responses, in gene expression, and/or in posttranslational maturation of lamin A (2). The familial partial lipodystrophy of the Dunnigan type (FPLD2) is due to few heterozygous substitutions in the carboxyl-terminal domain of A-type lamins, with the mutational hot spot located at residue R482. FPLD2 is characterized by partial lipoatrophy with progressive postpubertal loss of sc adipose tissue in limbs, abdomen, and trunk. Excess fat deposition can occur in face, neck, and intraabdominal regions. Other clinical features include muscular hypertro-

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phy predominant in calves, acanthosis nigricans, and, in women, polycystic ovary syndrome. The biological hallmarks are insulin resistance leading to diabetes and severe hypertriglyceridemia with a risk of acute pancreatitis (7, 8, 13, 14). In addition, atherosclerosis is precocious and severe (15), and liver steatosis is common (16). Complex genotype/phenotype relationships are observed in laminopathies. Signs of limb-girdle muscular dystrophy can be observed in the typical LMNA codon 482-associated FPLD2 (17), and lipoatrophy is part of the phenotype in mandibuloacral dysplasia (18) and Hutchinson-Gilford progeria (19). Moreover, mixed laminopathy phenotypes due to non-codon 482-associated mutations have been described, associating lipodystrophy and skeletal and/or cardiac dystrophic features (20 –22) or premature aging symptoms (23–26). Attenuated phenotypes of FPLD2 have been linked to p.R582H or p.S583L mutations affecting the lamin A isoform (27, 28). We are routinely sequencing the entire coding region of LMNA in patients referred for lipodystrophy and/or android body habitus, insulin resistance, or altered glucose tolerance. Between 2002 and 2006, 277 unrelated adults were screened. Seventeen patients were diagnosed with codon 482 heterozygous substitutions and 10 patients with non-codon 482 mutations. We previously reported one of these patients, presenting with a type A insulin resistance syndrome without clinical lipodystrophy (29). The clinical presentations of the nine additional patients also differed from that of typical FPLD2, because lipodystrophy was mild or absent in most cases. Subjects and Methods Subjects We studied nine patients with diabetes and/or lipodystrophy and non-codon 482 LMNA mutations. All the patients gave their written informed consent for these studies.

Phenotype and genotype characterization Subjects underwent a complete clinical evaluation. Metabolic investigations were performed after a 12-h fasting period. Glucose and insulin were measured at 0 and 120 min of a 75-g oral glucose tolerance test (OGTT). Total and segmental body fat was evaluated by whole-body dual-energy-x-ray absorptiometry (DEXA, Lunar model DPX; GE Medical Systems, Waukesha, WI). Quantification of sc and visceral fat was performed on abdominal and/or mid-thigh cross-sectional computed tomography (CT) scan or magnetic resonance imaging (MRI) using OsiriX Medical Imaging Software. Cardiac investigations included electrocardiogram and 24-h rhythm monitoring. LMNA sequencing was performed as previously described (13).

Cellular studies Primary fibroblast cultures were established by skin biopsy. EpsteinBarr virus-immortalized lymphoblastoid cells were derived from circulating lymphocytes. Immunofluorescence microscopy and Western blot analysis were performed as previously described (30). Lymphoblastoid cells were loaded onto polylysine-coated glass coverslips, fixed in 4% paraformaldehyde, and incubated for 30 min in 1% BSA and 1% Triton X-100 before staining. We used antibodies against lamin A/C (MAB-3211; Chemicon International Inc., Temecula, CA), lamin B (a generous gift from B. Buendia, Centre National de la Recherche Scientifique, Institut Jacques Monod, Universite´s Paris 6 et 7, Paris, France), and ␤-actin (A5441; Sigma-Aldrich, St. Quentin-Fallavier, France).

Decaudain et al. • Metabolic Laminopathies

Results Case reports

The patients’ main features are summarized in Tables 1 and 2. Patients were of Caucasian origin except patient 9. Figure 1 gives a schematic representation of the lamin A/C gene and proteins with the localization of the mutations (Fig. 1A) and shows the conservation of the mutated residues among species (Fig. 1B). Figure 2 shows pictures of patients 1 and 8 and mid-thigh and/or abdominal CT scan or MRI from control and patients. Patient 1 was referred for obesity with sc infiltration of hands and feet. Android fat distribution and calves hypertrophy were the main clinical features (Fig. 2, A and C). An incidentally discovered noninvasive pituitary microadenoma was shown to be nonfunctioning after hormonal investigations of all pituitary secretion axes. In particular, the nycthemeral variation of plasma cortisol (morning, 254 nmol/liter; evening, 27 nmol/liter), the 24-h urinary free cortisol (102 nmol), and the overnight 1-mg dexamethasone suppression test (cortisol after suppression, 11 nmol/liter) were normal. The insulin tolerance test, i.e. iv injection of 0.1 IU/kg insulin with multiple glucose sampling until 15 min and calculation of the coefficient of glycemic variation (DG)to-initial glycemia (D0) ratio (31), showed major insulin resistance (DG/D0 ⫽ 0.26, mean ⫾ sd for controls, 0.59 ⫾ 0.12) (31). The 53-yr-old mother of the patient was lean [body mass index (BMI) ⫽ 24 kg/m2], exhibited diffuse sc lipoatrophy sparing the face and muscular hypertrophy of the four limbs, and suffered from diabetes, hypertension, and mixed dyslipidemia since age 25. LMNA sequencing showed a heterozygous g.498C3 T substitution predicting a p.R28W mutation both in the proband and her mother but not in her 22-yr-old asymptomatic sister. Patient 2 received high insulin doses for diabetes diagnosed at age 23 and complicated with peripheral neuropathy, retinopathy, and hypertension. Physical examination revealed an android body habitus. Peripheral sc lipoatrophy of the four limbs contrasted with accumulation of truncal fat and proximal amyotrophy of the lower limbs with calves hypertrophy. The patient complained of joint and muscle pain affecting shoulders, pelvic girdle, and hands, and of frequent calf cramps. Electromyogram of the four limbs revealed proximal myogenic alterations and distal peripheral neuropathy. Cardiac investigations were normal except for a first degree atrioventricular block. After a 3-h insulin iv infusion in the fasting state, insulinemia reached 177 mU/ liter, whereas glycemia remained at 8.3 mmol/liter, showing a severe insulin resistance. LMNA sequencing showed a heterozygous g.960C3 T substitution predicting a p.L92F mutation. Sudden cardiac deaths occurred in her father at age 56 and her paternal grandfather, who bore a pace-maker, at age 64. Familial genetic screening could not be performed. Patient 3 presented with insulin-requiring diabetes from age 12, gradually complicated with retinopathy and neuropathy. She complained of chronic myalgia and articular pain in upper limbs (shoulders, elbows, wrists, and hands) and ankles since age 30. Physical examination revealed an android fat distribution without any sc lipoatrophy. Her mother exhibited android obesity and insulin-treated diabe-

Normal

Cardiological examination, ECG, rhythm monitoring

Calves hypertrophy, proximal lower limbs amyotrophy, cramps, joint and muscle pain

First-degree atrioventricular block

No, but android baldness Treated hypertension

Oligomenorrhea, one successful pregnancy

No

Treated hypertension Diffuse atherosclerosis, coronary and iliac angioplasties

–

120/70 Normal

Diffuse myalgias, articular pain

–

Normal

Calves hypertrophy, cramps, pelvic muscle weakness

Normal

Lower limbs pain and weakness

Diffuse atherosclerosis, coronary bypass at age 49

Calves hypertrophy, myalgias of lower limbs, no muscle weakness

110/70

No

Face, neck, hands, feet, pubis Polycystic ovary syndrome, one successful pregnancy after metformin therapy

80/1.68 28.3 99 Axillae, neck Four limbs

Treated hypertension

No

Three successful pregnancies

Diffuse

96/1.54 40.5 112 No No


No

Irregular and anovulatory cycles, infertility

–

Three pregnancies leading to miscarriages

No

No

65/1.67 23.3 85 No Lower limbs

Face, neck

90/1.70 31.1 103 No No

No

64/1.54 27 98 No No

Ischemic cardiopathy, rhythm and conduction disturbances (pacemaker, then defibrillator) Generalized muscular hypertrophy, cramps, myalgia Calves hypertrophy, perihumeral muscular atrophy


Severe

Polycystic ovary syndrome

Four–limbs, abdomen, and trunk Face

48/1.51 21.1 80

Normal

130/80

Face, neck, hands, feet Endometriosis with primary infertility requiring hysterectomy and bilateral ovariectomy at age 40 No

64/1.68 22.7 87 No Four limbs

Generalized muscular hypertrophy and diabetes in a brother and a sister Similar morphotype in her mother

Obesity and hypertension in her mother

Obesity in three siblings and three children

Diabetes in her deceased mother and brother

Diabetes in a maternal aunt and maternal grandmother

Mother: android obesity and diabetes; brother: insulin-resistant diabetes, myocardial infarction, and stroke at 54

Sudden cardiac death of her father (age 56) and paternal grandfather (age 64, despite pacemaker) 56/1.61 21.6 88 No Four limbs

F 53 Homozygous p.T655fsX49, exon 11, lamin A-specific C-terminal domain

Patient 9

F 56 Heterozygous p.H506D, exon 9, C-terminal domain

Patient 8

F 33 Heterozygous p.R439C, exon 7, C-terminal domain

Patient 7

F 57 Heterozygous p.L421P, exon 7, C-terminal domain, NLS

Patient 6

F 54 Heterozygous p.R399H, exon 7, C-terminal domain

Patient 5

M 60 Heterozygous p.S395, exon 7 C-terminal domain

Patient 4

F 61 Heterozygous p.L387V, exon 7, C-terminal domain

Patient 3

F 49 Heterozygous p.L92F, exon 1, rod domain, coil 1B

Patient 2

Age indicates the age at examination and diagnosis of laminopathy. ECG, Electrocardiogram; NLS, nuclear localization signal; –, no data

Calves hypertrophy

110/70

Blood pressure (mm Hg)

Muscular complaints and examination

Face

Face, neck, hands, and feet Oligomenorrhea, one successful pregnancy

Hirsutism

Gynecological history

Fat accumulation

84/1.66 30.5 101 Axillae, neck Slight, lower limbs

Partial lipoatrophy, diabetes, and hypertension in the mother with the same LMNA mutation

Familial antecedents

Weight (kg)/height (m) BMI (kg/m2) Waist circumference (cm) Acanthosis nigricans Clinical lipoatrophy

F 24 Heterozygous p.R28W, exon 1, N-terminal domain

Sex Age (yr) LMNA mutation

Patient 1

TABLE 1. Main clinical features of patients

Decaudain et al. • Metabolic Laminopathies J Clin Endocrinol Metab, December 2007, 92(12):4835– 4844 4837

–

–

–

21.8/26.8 22.7/14.9

Normal

4 –

TG, 3.4; Total/HDL-chol, 4.8/1.1, under statins

8.5 –

Diabetes, insulin 1.4 U/ kg䡠d

Patient 4

HbA1c, Glycated hemoglobin; HDL-chol, HDL-cholesterol; N, normal; TG, triglycerides; –, no data.

Treated hypothyroidism

–

–

35.2/37.5 36.7/30.5

Normal

– –

TG, 1.5; Total/HDLchol, 5.3/1.5, under fibrates

– –

Diabetes, insulin 1.2 U/kg䡠d

Patient 3

–

–

Impedancemetry: 10.8% of fat (N, 30.3 ⫾ 1.5) –

Normal

16.2 –

TG, 17.1; Total/ HDL-chol, 7.8/1.4, under fibrates

8.5 –


Patient 5

Multinodular goiter, obstructive sleep apnea

–

–

41.7/42.7 42/44.1

–

Abdomen, no sc lipoatrophy; increased visceral fat Liver steatosis on abdominal MRI

–

Impaired glucose tolerance

Patient 7

Patient 8

Treated hypothyroidism

Normal on ultrasound examination

–

15.1/18.8 12/11.1

Impaired fasting glucose normalized under diet 7.1 5.4 5.7 – T0, 4.6/21; T120 T0, 4.8/14; min, 11/117 T120 min, 5.1/ 56 TG, 2.9; Total/ TG, 1.8; Total/ TG, 1.2; Total/ HDL-chol, HDL-chol, HDL-chol, 4.4/1.0, under 5.2/1.2 3.9/1.1, under statins statins 69 23.2 2.2 – Androstenedione, – 14.9 (n ⬍ 10.7); Testosterone, 3.1 (n ⬍ 2.8) Normal Normal Normal

Diabetes treated with metformin

Patient 6

–

–

–

15.6/17.4 14.8/9.5

Normal

– Testosterone, 7.3 (N ⬍ 2.8)

TG, 4.6; Total/ HDL-chol, 3.3/0.4

8 –

Diabetes, insulin 4 U/kg䡠d

Patient 9


Other features

Liver hypertrophy and steatosis on ultrasound examination Bilateral cataract at age 42

Liver hypertrophy and steatosis on ultrasound examination Nonsecreting pituitary microadenoma

Liver imagery

–

Fatty degeneration Abdomen (L4), mild of gluteus and thigh sc lipoatrophy; adductors increased visceral fat

20.1/20.3 26/16.7

Normal

7 –

MRI

Liver enzymes DEXA: % fat Whole body/ trunk Upper/lower limbs

20 Androstenedione, 12.8 (N ⬍ 10.7); Testosterone, 3.0 (N ⬍ 2.8) Normal

TG, 2.4; Total/HDLchol, 5.2/2.2, under statins

TG, 6; Total/HDLchol: 4.8/0.74

Serum leptin (ng/ml) Hormonal investigations (nmol/liter)

10.5 –

5.4 T0, 4.8/48; T120 min, 10/-

HbA1c (%) OGTT: glucose (mmol/liter)/ insulin (mU/liter) Lipid profile (mmol/liter)


Impaired glucose tolerance

Patient 2

Glucose tolerance status and treatment

Patient 1

TABLE 2. Main biological and imaging features of patients

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FIG. 1. A, Schematic representation of the LMNA gene and lamins A/C proteins with the localization of mutations; B, amino acid sequences alignment for lamin A/C from various species. The residues affected by the mutations described in this study are shown in bold. The Swiss Prot accession numbers are P02545 and P48678 for human and mouse lamin A/C, respectively, and P48679, P13648, and P11048 for rat, chicken, and Xenopus laevis lamin A, respectively.

tes, and her brother, treated with 160 U/d insulin for diabetes since age 40, had presented a coronary heart disease and a stroke in his fifth decade. LMNA sequencing showed a heterozygous g.3135C3 G substitution predicting a p.L387V mutation. Familial genetic screening could not be performed. Patient 4 had an insulin-resistant diabetes diagnosed at age 42 and complicated with retinopathy, nephropathy, and peripheral neuropathy. He was suffering from hypertension and severe and diffuse atherosclerosis since age 46, with coronary heart disease, carotid stenosis, and lower limbs ischemia. Physical examination revealed android obesity with fat accumulation in the neck and face, but without any obvious clinical sc lipoatrophy. However, DEXA revealed a slight lower limbs lipoatrophy, and serum leptin level was low. Muscular examination and cardiac investigations were normal. LMNA sequencing showed a heterozygous g.3160C3 T substitution predicting a p.S395S mutation in the proband but not in his 33-yr-old asymptomatic son. Patient 5 was diagnosed with insulin-resistant diabetes at age 32. Chronic hyperglycemia led to retinopathy, peripheral neuropathy, and renal failure. A severe hypertriglyceridemia was maintained despite lipid-lowering therapies. She had a diffuse atherosclerosis, and a coronary artery bypass was required at age 49. Physical examination revealed android fat distribution, lipoatrophy of lower limbs and calves hypertrophy without any muscle weakness. LMNA sequencing showed a heterozygous g.3172G3 A substitution predicting a p.R399H mutation. Her mother and one of her two brothers had diabetes and died several years ago. Her 50-yr-old second brother and 49-yr-old maternal cousin were not diabetic and did not carry the mutation.

Patient 6 was referred for morbid android obesity that progressively worsened since age 20, with diabetes diagnosed at age 55. She was also suffering from hypertension complicated with left ventricular hypertrophy. She underwent three successful pregnancies, but birth weights were all above 4 kg. Clinical examination did not report any lipoatrophy, in accordance with DEXA results and serum leptin level. Lower limbs pain and weakness with areflexia were observed. LMNA sequencing showed a heterozygous g.3238T3 C substitution predicting a p.L421P mutation in the nuclear localization signal domain of the protein. Familial genetic screening could not be performed. Patient 7 was referred at age 33 for fat accumulation in face, neck, upper thorax, and pubic areas that progressively developed since puberty. She complained of sc painful pseudoedema of hands and feet, of muscle cramps in the lower limbs, and of pelvic muscles weakness. Physical examination showed marked sc lipoatrophy of the four limbs and calves hypertrophy. OGTT revealed impaired glucose tolerance with hyperinsulinemia. LMNA sequencing showed a heterozygous g.3290C3 T substitution predicting a p.R439C mutation. The patient refused to inform her family about her disease, so no familial genetic screening could be performed. Patient 8 was referred at age 56 for subclavicular fat accumulation. She underwent plastic surgery for double-chin reduction at age 30 and for several small sc lipomas of limbs and adrenal surgery for angiomyolipoma at age 50. Lipodystrophy, with loss of sc fat in the four limbs contrasting with cervicofacial fat accumulation, was noticed since puberty. Clinical examination also revealed enlarged hands and

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FIG. 2. A, Photograph from patient 1 bearing a p.R28W heterozygous LMNA mutation, showing an accumulation of facial and cervical fat, a slight lipoatrophy affecting lower limbs but not abdomen, a muscular hypertrophy of calves, and thick hands with broad, infiltrated, and spindle-shaped fingers; B, photograph from patient 8 bearing a p.H506D heterozygous LMNA mutation, showing an accumulation of fat in the supraclavicular area (a liposuction of the double chin was performed 26 yr ago) and perihumeral muscular atrophy with lipoatrophy of upper limbs; C, thigh and/or abdominal CT scans or MRI from control and LMNA-mutated women, as indicated. Adipose tissue appears in dark gray on CT scans and in white on MRI. Quantification of percent adipose tissue in abdominal L4 and in mid-thigh regions, calculated as described by Al-Attar et al. (42), shows that sc lipoatrophy is absent or markedly less severe in patients with LMNA non-codon 482-associated mutations than in those with codon 482-associated mutations. In addition, MRI showed a fatty degeneration of glutei and adductor muscles in patient 1.

feet and calves hypertrophy contrasting with perihumeral muscular atrophy (Fig. 2B). Impaired fasting glucose (6.8 mmol/liter) was controlled by diet and mixed dyslipidemia by statins. DEXA and serum leptin measurement confirmed the decreased amount of fat. LMNA sequencing showed a heterozygous g.4061C3 G substitution predicting a p.H506D mutation. Her mother was described as having the same morphotype, but familial genetic screening could not be performed. Patient 9 was an African-Creole woman originating from the Reunion Island, followed for an insulin-requiring diabetes. Her medical history revealed muscular hypertrophy since age 11, polycystic ovarian syndrome diagnosed at age 22, diabetes, hypertension, and hypertriglyceridemia since age 23, and several myocardial infarctions since age 43. Cardiac rhythm and conduction disturbances have required a pacemaker and then a defibrillator implantation for ventricular tachycardia at age 52. Clinical examination at age 53 revealed sc severe lipoatrophy of abdomen, trunk, and limbs contrasting with a round face and generalized muscular appearance. Muscular strength was normal, but she complained of cramps and myalgia. Creatine phosphokinase measurements were high (400 – 600 IU/liter) in the absence

of lipid-lowering treatment. Echocardiography revealed a dilated cardiomyopathy with a left ejection fraction of 25– 30%. LMNA sequencing showed a homozygous G insertion after nucleotide 5670 (g.5670_5671insG) predicting 48 aberrant amino acids to replace the last 10 carboxyl-terminal residues of lamin A (p.T655fsX49 frameshift mutation). No parental consanguinity was known. Among the six siblings of the patient, one brother and one sister (50 and 52 yr old) were described with generalized muscular hypertrophy and diabetes but refused genetic analyses. Another brother, also very muscular, died at age 48 of unknown cause. The proband died at age 54 after a paroxysmal arrhythmia, despite her cardioverter-defibrillator. Molecular studies

Sequencing of the LMNA exons 1, 7, 9, and 11 in DNA from 200 unrelated Caucasian controls did not reveal any of the described mutations. We have previously sequenced LMNA exon 11 in DNA from 194 Creole subjects with African ancestry from the Reunion Island (29), and we did not find the p.T655fsX49 mutation.



Comparisons between data from patients with either LMNA non-codon 482 mutations or codon 482 mutations

We compared the data from the nine patients presented here and from the previously described LMNA G602Smutated patient (29) with those from 11 women of similar age at examination (mean age 47 and 39 yr, respectively; P ⫽ 0.18), followed in our medical departments for typical FPLD2 due to codon 482 LMNA heterozygous mutations. As depicted in Table 3, patients with non-codon 482 LMNA mutations were significantly older at diagnosis and had higher BMI, serum leptin, waist circumference, and percent total and segmental body fat than patients with codon 482 mutations. Prevalence of glucose tolerance alterations and serum levels of total cholesterol and triglycerides were not different between the two groups of patients, whereas high-density lipoprotein (HDL)-cholesterol was lower in patients with codon 482 mutations. The main phenotypic difference between the two groups was the clinical abdominal sc lipoatrophy that affected only one of the patients with non-codon 482 mutations but all patients with codon 482 mutations (Table 3). Quantification of adipose tissue on mid-thigh and/or L4 abdominal CT scan or MRI confirmed that sc lipoatrophy was absent or very mild in patients with non-codon 482 mutations, compared with patients with codon 482 mutations (Fig. 2C). Cardiac conduction disturbances were not found in patients with codon 482 mutations but affected two patients with noncodon 482 mutations. Menstrual irregularities and/or hirsutism and muscular signs were frequently observed in both groups of patients.

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Cellular studies (Fig. 3)

Fifteen to 25% of fibroblasts issued from LMNA-mutated patients and 5% of control cells showed dysmorphic nuclei with herniations at passage 6. LMNA-mutated lymphoblastoid cells showed 23–31% of blebbing nuclei, compared with 9% of control cells. In addition, heterogeneous staining of lamin A/C and absent lamin B staining, typical of laminopathies, was observed in 20 – 40% of dysmorphic LMNAmutated nuclei but no control nuclei. We did not observe significant qualitative or quantitative differences in nuclear dysmorphism according to the different LMNA mutations. Western blot analysis of lymphoblastoid cell extracts showed that mutated lamin A from patient 9 had a retarded electrophoretic mobility, in accordance with the increased molecular weight of the protein predicted by the LMNA g.5670_5671insG homozygous mutation. Discussion

Among laminopathies, FPLD2 displays specific hallmarks. Postpubertal onset of sc lipoatrophy affecting the four limbs and abdomen but sparing face and neck, calves hypertrophy, hypertriglyceridemia, and insulin resistance, more severe in women than in men (13, 32, 33), are typical features present in most cases. The observations reported in this paper show that noncodon 482 LMNA mutations, located in the three domains of type A lamins, can be detected in patients presenting with a severe metabolic syndrome. All nine patients, and our previously described case (29), fulfilled the International Diabetes Federation diagnosis criteria for metabolic syndrome

TABLE 3. Comparisons of the clinical and biological features among the 10 patients with metabolic laminopathies due to LMNA noncodon 482 mutations and 11 women with FPLD2 (codon 482 mutations) Patients with metabolic laminopathy

Age at diagnosis of laminopathy (yr) BMI (kg/m2) Waist circumference (cm) Patients with acanthosis nigricans Patients with clinical abdominal sc lipoatrophy Women with oligomenorrhea and/or hirsutism Patients with hypertension Patients with cardiac conduction disturbances Patients with calves hypertrophy Patients with myalgia and/or muscular weakness Patients with articular pain Patients with sc lipomas Glucose tolerance Serum triglycerides (mM) Serum total cholesterol (mM) Serum HDL-cholesterol (mM) Serum leptin (ng/ml) DEXA % total body fat % trunk fat % upper limbs fat % lower limbs fat

47 ⫾ 12 26.9 ⫾ 4.6 93.6 ⫾ 9.0 3/10 1/10 5/9 5/10 2/10 7/10 5/10 2/10 1/10 Abnormal in 10/10 (diabetes in 6, IGT in 3, IFG in 1 patient) 4.3 ⫾ 2.9 4.9 ⫾ 0.8 1.2 ⫾ 0.3 19.3 ⫾ 13.6 25.4 ⫾ 8.3 28.2 ⫾ 8.5 25.5 ⫾ 8.0 21.4 ⫾ 9.6

Patients with FPLD2

P value

37 ⫾ 11 23.4 ⫾ 1.7 79.6 ⫾ 3.2 8/11 11/11 7/11 6/11 0/11 10/11 6/11

0.05 ⬍0.05 ⬍0.003 0.06 ⬍0.001 0.54 0.59 0.21 0.26 0.59

7/11 5/11 Abnormal in 10/11 (diabetes in 10, normal in 1 patient) 5.5 ⫾ 2.2 5.1 ⫾ 1.1 0.8 ⫾ 0.2 5.4 ⫾ 2.6 14.7 ⫾ 3.6 18.1 ⫾ 2.8 10.9 ⫾ 4.2 9.2 ⫾ 3.7

0.06 0.09 0.55 0.24 0.38 ⬍0.04 ⬍0.04 ⬍0.02 ⬍0.02 ⬍0.005 ⬍0.02

All data are mean ⫾ SEM. IFG, Impaired fasting glucose; IGT, impaired glucose tolerance. P was calculated using Student’s t test for continuous variables and Fisher’s exact test for qualitative data.

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FIG. 3. Immunofluorescence studies in cultured fibroblasts (A) and lymphoblastoid cells (B) from LMNA-mutated and control patients. Cultured skin fibroblasts were issued from patients 2, 3, 5, 6, and 8 (LMNA mutations p.L92F, p.L387V, p.R399H, p.L421P, and p.H506D, respectively), from two women with typical FPLD2 due to the LMNA p.R482W mutation (aged 43 and 54 yr), and from two nonobese, nondiabetic control women (aged 20 and 33 yr). Lymphoblastoid cells were obtained from patients 4 (LMNA p.S395L) and 9 (LMNA p.T655fsX49), from the same FPLD2 women, and from a nonobese, nondiabetic control woman. Nuclear staining with diamidino-2-phenylindole hydrochloride (DAPI) (DNA, blue) and with antibodies directed against lamin A/C (red) and lamin B (green) is shown in representative photographs. Note the nuclear herniations in LMNA-mutated fibroblasts. Lamin A/C staining was heterogeneous and lamin B staining reduced or absent in 20 – 40% of nuclear blebs or poles in LMNA-mutated cells but not in control cells. Nuclear dysmorphisms (one or several nuclear blebs) were quantified in 200 cells for each case, at passage 6 for fibroblasts. C, Western blot analysis of lymphoblastoid cells extracts. ␤-Actin was used as a loading control. The apparent molecular weight of mutated lamin A from patient 9 was higher than that of wild-type lamin A.

(34) and lacked some specific signs observed in typical LMNA codon 482-associated FPLD2. Indeed, no clinical sc lipoatrophy was detectable in three of our nine patients, with DEXA showing global obesity without any decrease in segmental fat in one of them. Among the six other patients, only one presented with the abdominal sc lipoatrophy that characterizes typical FPLD2. Only one of the seven tested patients had low serum leptin levels. Five patients were not primarily referred for altered fat distribution, but for insulin-resistant diabetes. Patients with non-codon 482 mutations had significantly higher BMI and waist circumference than patients with codon 482 mutations. Metabolic alterations were prominent; insulin resistance, evidenced by very high insulin requirements or hyperinsulinemia, was present in all but one patient and hypertriglyceridemia with low HDL-cholesterol in all of them. Other features commonly related to both metabolic syndrome and FPLD2 were found in some patients with non-codon 482 mutations: imaging evidence for liver steatosis, precocious atherosclerosis, menstrual irregularities, and hirsutism in women. Because the phenotype of these patients was not typical of FPLD2, the diagnosis of laminopathy was delayed. However, although lipodystrophy was less severe than in typical FPLD2, several additional signs justified the search for LMNA mutations. Calves hypertrophy and/or myalgia, cramps, or muscle weakness was present in all patients but one, and cardiac conduction disturbance was reported in two patients and was lethal in one of them despite a previous defibrillator implantation, indicating the importance of careful cardiac follow-up. Interest-

ingly, several patients presented with articular pain. Alterations in joint mobility with arthritic changes are also observed in LMNA-linked progeroid syndromes (19, 25) and in FPLD2. Together with hand and foot discomfort due to fat infiltration, muscular cramps, myalgia, and diabetic neuropathy, laminopathies can be very painful diseases. We observed the progressive appearance of small sc lipomas of limbs, trunk, and abdomen in one patient with non-codon 482 mutations and in five typical FPLD2 patients; the prevalence of this sign needs to be evaluated in laminopathies. Three of our nine patients presented with thyroid abnormalities, which have also been reported in FPLD2, although the link with LMNA mutations could not be proved (35). Familial molecular analyses could not be performed in most of our cases. The familial cosegregation of the disease and the genetic trait was shown only for patient 1. However, these LMNA mutations were not found in 200 unrelated controls of the same ethnicity, and the mutated residues are, for most of them, strongly conserved among species (Fig. 1B). In addition, we found the specific nuclear alterations linked to laminopathies, with a similar percentage of affected cells, in cultured skin fibroblasts or lymphoblastoid cells from our patients (Fig. 3) (9 –11, 30, 36). Furthermore, residues at positions 28, 387, 395, and 399 are located within or in the very close vicinity of positively charged clusters that are thought to allow the head-to-tail association of two dimers through strong electrostatic interactions (37). Nevertheless, our study could not provide familial or cellular data giving evidence for the pathogenic effect of the p.R439C mutation, although a


laminopathy was strongly suggested by the patient’s body fat distribution, metabolic and hormonal alterations, and muscle signs. The p.R28W mutation had already been described in a family with a mixed phenotype of lipodystrophy and DCM-CD (21). Our observation of patient 1 shows that this mutation can lead to metabolic alterations without any cardiac abnormalities, at least at presentation. Recently, the first case of diabetes and partial lipodystrophy linked to a mutation in the rod domain of lamins A/C was described (38). The observation of patient 2 adds additional evidence that alterations in the rod domain of type A lamins, already known to be involved in cardiac and neuromuscular diseases, can also underlie metabolic laminopathies. The novel LMNA p.L421P mutation affects the nuclear localization signal domain (NLS) of lamins A/C. Only one mutation had been previously reported in this domain (39) and was associated with FPLD2, although the phenotype was not detailed. LMNA p.L421P-mutated fibroblasts showed an exclusively intranuclear staining of lamins A/C, suggesting that the trafficking of the protein was not impaired. We report for the first time a homozygous LMNA p.T655fsX49 mutation with a severe phenotype associating partial lipodystrophy, severe metabolic disturbances, and DCM-CD. LMNA mutations in exon 11 were previously linked to very diverse laminopathies, including type A insulin resistance (29), classical or mild FPLD2 (13, 27, 28), muscular skeletal or cardiac dystrophies (40), progeria (10, 11), and restrictive dermopathy (12). Interestingly, the p.T655fsX49 mutation predicts that the farnesylation of prelamin A, which normally occurs in the CSIM carboxyl-terminal motif of the protein, could not be possible. Hutchinson-Gilford progeria is also thought to result from the accumulation of an immature lamin A, but it remains farnesylated (2). Our patient did not show any clinical sign of premature aging, adding additional evidence for the specific involvement of the farnesyl motif of immature lamin A in progeria. A correlation of LMNA mutation positions, upstream or downstream of the NLS sequence, and organ system involvement was identified by Hegele (41). Class 1 laminopathies included those with neuromuscular or cardiac involvement and overlapping syndromes, whereas class 2 laminopathies included partial lipodystrophy and accelerated aging syndromes. Patients 1 and 2 with overlapping laminopathic signs and mutations affecting the amino-terminal or rod domain of the protein on the one side, and patients 7 and 8 with a phenotype close to that of FPLD2 including muscle alterations (17) and mutations downstream of the NLS sequence on the other side, clearly fit the class 1 and 2 laminopathies, respectively. However, our study shows that LMNA mutations in exon 7, which encoded amino acids located upstream and downstream of the NLS sequence, and in exon 11, are more difficult to classify. Pathophysiological studies linking these A-type lamin domains to such various phenotypes are of particular interest. In conclusion, we show here that metabolic syndrome, without obvious clinical lipoatrophy, can be the prominent manifestation revealing LMNA mutations. Interestingly, Atype lamin alterations are different from the typical FPLD2linked codon 482 mutations and can affect all the protein


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domains. We suggest that patients with insulin resistance syndromes should be investigated with DEXA and/or crosssectional CT scan or MRI at the abdominal L4 and thigh levels and should be screened for mutations in LMNA when a subclinical lipodystrophy is evidenced or when muscular hypertrophy, myalgias, or weakness are present. A careful cardiac follow-up is required for all patients with LMNA mutations. The prevalence of metabolic laminopathies, which affected some small percentage of our patients with severe metabolic syndrome, needs to be more precisely evaluated, and additional studies are required to explain the link between LMNA mutations and insulin resistance. Acknowledgments We are grateful to Yves Chre´tien for editorial assistance; to Dominique Pontoire and Claudine Treyz for immortalization of patients’ lymphocytes in the Banque de Cellules, Groupe Hospitalier Cochin, Assistance Publique-Hoˆpitaux de Paris, directed by Prof. Jamel Chelly; and to Dr. Ce´line Droumaguet, Service d’Endocrinologie, directed by Prof. Philippe Chanson, Hoˆpital Biceˆtre, Assistance Publique-Hoˆpitaux de Paris, for providing one of the control abdominal MRI. Received March 22, 2007. Accepted August 13, 2007. Address all correspondence and requests for reprints to: C. Vigouroux, Faculte´ de Me´decine Universite´ Pierre et Marie Curie-Paris 6, INSERM U680, 27, rue Chaligny, 75571 Paris Cedex 12, France. E-mail: [email protected]. This work was supported by INSERM and European Union’s FP6 Life Science, Genomics, and Biotechnology for Health LSHM-CT-2005018690 and by grants from the Association de Langue Française pour l’Etude du Diabe`te et des Maladies Me´taboliques (ALFEDIAM) (to C.V.), and the Fondation pour la Recherche Me´dicale (to A.D. and B.D.). Disclosure Statement: The authors have nothing to declare.

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