Compound heterozygosity for mutations in LMNA in a patient with a myopathic and lipodystrophic mandibuloacral dysplasia type A phenotype

CONTEXT: Mandibuloacral dysplasia type A (MADA; OMIM 248370) is a rare progeroid syndrome characterized by dysmorphic craniofacial and skeletal features, lipodystrophy, and metabolic complications. Most Italian patients carry the same homozygous missense mutation (p.R527H) in the C-terminal tail domain of the LMNA gene, which encodes lamin A/C, an intermediate filament component of the nuclear envelope. OBJECTIVE: The objective of the study was to identify novel LMNA mutations in individuals with clinical characteristics (bird-like facies, mandibular and clavicular hypoplasia, acroosteolysis, lipodystrophy, alopecia) observed in other well-known patients. DESIGN: The LMNA gene was sequenced. Functional properties of the mutant alleles were investigated. PATIENT: We report a 27-yr-old Italian woman showing a MADA-like phenotype. Features include a hypoplastic mandible, acroosteolysis, pointed nose, partial loss of sc fat, and a progeric appearance. Due to the absence of clavicular dysplasia and normal metabolic profiles, generally associated with muscle hyposthenia and generalized hypotonia, this phenotype can be considered an atypical laminopathy. RESULTS: We identified a patient compound heterozygote for the p.R527H and p.V440M alleles. The patient's cells showed nuclear shape abnormalities, accumulation of pre-lamin A, and irregular lamina thickness. Lamins A and C showed normal expression and localization. The electron microscopy detected heterochromatin defects with a pattern similar to those observed in other laminopathies. However, chromatin analysis showed a normal distribution pattern of the major heterochromatin proteins: heterochromatin protein-1beta and histone H3 methylated at lysine 9. CONCLUSIONS: The clinical and cellular features of this patient show overlapping laminopathy phenotypes that could be due to the combination of p.R527H and p.V440M alleles.     

Blocking protein farnesyltransferase improves nuclear blebbing in mouse fibroblasts with a targeted Hutchinson-Gilford progeria syndrome mutation

Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a "CAAX protein" that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by chi2 statistic). These studies suggest a possible treatment strategy for HGPS.     

Blocking protein farnesyltransferase improves nuclear shape in fibroblasts from humans with progeroid syndromes

Defects in the biogenesis of lamin A from its farnesylated precursor, prelamin A, lead to the accumulation of prelamin A at the nuclear envelope, cause misshapen nuclei, and result in progeroid syndromes. A deficiency in ZMPSTE24, a protease involved in prelamin A processing, leads to prelamin A accumulation, an absence of mature lamin A, misshapen nuclei, and a lethal perinatal progeroid syndrome: restrictive dermopathy (RD). Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutant prelamin A that cannot be processed to lamin A. The hallmark cellular abnormality in RD and HGPS is misshapen nuclei. We hypothesized that the farnesylation of prelamin A is important for its targeting to the nuclear envelope in RD and HGPS and that blocking farnesylation would ameliorate the nuclear shape abnormalities. Indeed, when RD fibroblasts were treated with a farnesyltransferase inhibitor (FTI), prelamin A was partially mislocalized away from the nuclear envelope, and the frequency of nuclear shape abnormalities was reduced (P < 0.0001). A FTI also mislocalized prelamin A and improved nuclear shape in Zmpste24-deficient mouse embryonic fibroblasts (P < 0.0001) and improved nuclear shape in human HGPS fibroblasts (P < 0.0001). Most remarkably, a FTI significantly improved nuclear shape in two fibroblast cell lines from atypical progeria patients with lamin A missense mutations in the absence of prelamin A accumulation (P = 0.0003 and P < 0.0001). These findings establish a paradigm for ameliorating the most obvious cellular pathology in lamin-related progeroid syndromes and suggest a potential strategy for treating these diseases.     

Hutchinson-Gilford progeria mutant lamin A primarily targets human vascular cells as detected by an anti-Lamin A G608G antibody

Hutchinson-Gilford progeria syndrome (HGPS; Online Mendelian Inheritance in Man accession no. 176670) is a rare disorder that is characterized by segmental premature aging and death between 7 and 20 years of age from severe premature atherosclerosis. Mutations in the LMNA gene are responsible for this syndrome. Approximately 80% of HGPS cases are caused by a G608 (GGC-->GGT) mutation within exon 11 of LMNA, which elicits a deletion of 50 aa near the C terminus of prelamin A. In this article, we present evidence that the mutant lamin A (progerin) accumulates in the nucleus in a cellular age-dependent manner. In human HGPS fibroblast cultures, we observed, concomitantly to nuclear progerin accumulation, severe nuclear envelope deformations and invaginations preventable by farnesyltransferase inhibition. Nuclear alterations affect cell-cycle progression and cell migration and elicit premature senescence. Strikingly, skin biopsy sections from a subject with HGPS showed that the truncated lamin A accumulates primarily in the nuclei of vascular cells. This finding suggests that accumulation of progerin is directly involved in vascular disease in progeria.     

Nucleoplasmic localization of prelamin A: implications for prenylation-dependent lamin A assembly into the nuclear lamina.

The synthesis of the nuclear lamina protein lamin A requires the prenylation-dependent processing of its precursor protein, prelamin A. Unlike p21ras, which undergoes similar initial posttranslational modifications, maturation of lamin A results in the proteolytic removal of the prenylated portion of the molecule. We have used an in vitro prenylation system to demonstrate the nature of the prenyl substituent on prelamin A to be a farnesyl group. Further, the in vitro farnesylation of prelamin A requires an intact cysteine-aliphatic-aliphatic-other (CAAX) amino acid sequence motif at its carboxyl terminus. The effect of blocking the prenylation of prelamin A on its localization and assembly into the nuclear lamina was investigated by indirect immunofluorescence. Expression of wild-type prelamin A in lovastatin-treated cells showed that nonprenylated prelamin A accumulated as nucleoplasmic particles. Upon addition of mevalonate to lovastatin-treated cells, the wild-type lamin A was incorporated into the lamina within 3 hr. Expression of a mutant lamin A in which the carboxyl-terminal 21 amino acids were deleted resulted in a lamin molecule that was directly assembled into the lamina. These results indicate that the carboxyl-terminal peptide of prelamin A blocks its proper assembly into the nuclear lamina and that the prenylation-initiated removal of this peptide can occur in the nucleus.     

Genetic and phenotypic heterogeneity in patients with mandibuloacral dysplasia-associated lipodystrophy

Mandibuloacral dysplasia (MAD) is a phenotypically heterogeneous, rare autosomal recessive disorder characterized by mandibular and clavicular hypoplasia, acroosteolysis, delayed closure of cranial sutures, joint contractures, and mottled cutaneous pigmentation. Patients with MAD develop two patterns of lipodystrophy: type A pattern, with loss of sc fat from the extremities and normal or slight excess in the neck and truncal regions; and type B pattern, with a more generalized loss of sc fat involving the face, trunk, and extremities. Recently, affected patients from five consanguineous Italian pedigrees with partial lipodystrophy (type A) were reported to have a homozygous R527H mutation in LMNA (lamin A/C) gene. We carried out mutational analysis of LMNA in affected patients from six pedigrees. Affected patients from two pedigrees with type A lipodystrophy had the homozygous R527H mutation in LMNA. The other four affected subjects who had type B lipodystrophy did not have any mutation in the exons and splice site junctions of LMNA; RNA extracted from lymphoblasts of two of these patients also revealed normal sequence. In these four subjects, sequencing of other known genes implicated in lipodystrophies, i.e. AGPAT2, Seipin, and PPARG also revealed no substantial alterations. We conclude that MAD is a genetically and phenotypically heterogeneous disorder. Besides LMNA gene, other as yet unmapped loci could be linked to MAD.     

The neonatal progeroid syndrome (Wiedemann–Rautenstrauch): A model for the study of human aging?

The Wiedemann-Rautenstrauch syndrome (WRS) characterises a premature aging syndrome in which several features of human aging are apparent at birth therefore allowing their grouping as a neonatal progeroid condition. This differentiates WRS from other progeroid entities such as Hutchinson-Gilford progeria syndrome (HGPS) in which characteristics of premature aging become apparent some time after birth. The etiology of WRS remains unknown. Some studies have observed an autosomal recessive mode of inheritance. Several studies analysing telomere length and lamin A gene have not revealed any alterations. However, mutations in LMNA have been reported in several other atypical progeroid syndromes. Based on these observations, several hypothesis could be withdrawn concerning the etiology of WRS. The study of genes associated with lamin A metabolism, such as Zmpste24, and the metabolic pathways associated with insulin, such as protein kinase B or AKT, are of particular interest. We believe that WRS characteristics indicate that discovery of the gene and the metabolic pathway associated with this syndrome will most likely lead to new knowledge about the physiopathology of human aging.     

A new clinical condition linked to a novel mutation in lamins A and C with generalized lipoatrophy,insulin-resistant diabetes,disseminated leukomelanodermic papules,liver steatosis,and cardiomyopathy

A-Type lamins, arising from the LMNA gene, are intermediate filaments proteins that belong to the lamina, a ubiquitous nuclear network. Naturally occurring mutations in these proteins have been shown to be responsible for several distinct diseases that display skeletal and/or cardiac muscle or peripheral nerve involvement. These include familial partial lipodystrophy of the Dunnigan type and the mandibuloacral dysplasia syndrome. The pathophysiology of this group of diseases, often referred to as laminopathies, remains elusive. We report a new condition in a 30-yr-old man exhibiting a previously undescribed heterozygous R133L LMNA mutation. His phenotype associated generalized acquired lipoatrophy with insulin-resistant diabetes, hypertriglyceridemia, hepatic steatosis, hypertrophic cardiomyopathy with valvular involvement, and disseminated whitish papules. Immunofluorescence microscopic analysis of the patient's cultured skin fibroblasts revealed nuclear disorganization and abnormal distribution of A-type lamins, similar to that observed in patients harboring other LMNA mutations. This observation broadens the clinical spectrum of laminopathies, pointing out the clinical variability of lipodystrophy and the unreported possibility of hypertrophic cardiomyopathy and skin involvement. It emphasizes the fact that the diagnosis of genetic alterations in A-type lamins requires careful and complete clinical and morphological investigations in patients regardless of the presenting signs.     

Inhibiting farnesylation of progerin prevents the characteristic nuclear blebbing of Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder that is characterized by dramatic premature aging and accelerated cardiovascular disease. HGPS is almost always caused by a de novo point mutation in the lamin A gene (LMNA) that activates a cryptic splice donor site, producing a truncated mutant protein termed "progerin." WT prelamin A is anchored to the nuclear envelope by a farnesyl isoprenoid lipid. Cleavage of the terminal 15 aa and the farnesyl group releases mature lamin A from this tether. In contrast, this cleavage site is deleted in progerin. We hypothesized that retention of the farnesyl group causes progerin to become permanently anchored in the nuclear membrane, disrupting proper nuclear scaffolding and causing the characteristic nuclear blebbing seen in HGPS cells. Also, we hypothesized that blocking farnesylation would decrease progerin toxicity. To test this hypothesis, the terminal CSIM sequence in progerin was mutated to SSIM, a sequence that cannot be farnesylated. SSIM progerin relocalized from the nuclear periphery into nucleoplasmic aggregates and produced no nuclear blebbing. Also, blocking farnesylation of authentic progerin in transiently transfected HeLa, HEK 293, and NIH 3T3 cells with farnesyltransferase inhibitors (FTIs) restored normal nuclear architecture. Last, treatment of both early- and late-passage human HGPS fibroblasts with FTIs resulted in significant reductions in nuclear blebbing. Our results suggest that treatment with FTIs represents a potential therapy for patients with HGPS.     

Abolishing the prelamin A ZMPSTE24 cleavage site leads to progeroid phenotypes with near-normal longevity in mice

Prelamin A is a farnesylated precursor of lamin A, a nuclear lamina protein. Accumulation of the farnesylated prelamin A variant progerin, with an internal deletion including its processing site, causes Hutchinson–Gilford progeria syndrome. Loss-of-function mutations in ZMPSTE24, which encodes the prelamin A processing enzyme, lead to accumulation of full-length farnesylated prelamin A and cause related progeroid disorders. Some data suggest that prelamin A also accumulates with physiological aging. Zmpste24^−/− mice die young, at ∼20 wk. Because ZMPSTE24 has functions in addition to prelamin A processing, we generated a mouse model to examine effects solely due to the presence of permanently farnesylated prelamin A. These mice have an L648R amino acid substitution in prelamin A that blocks ZMPSTE24-catalyzed processing to lamin A. The Lmna^L648R/L648R mice express only prelamin and no mature protein. Notably, nearly all survive to 65 to 70 wk, with ∼40% of male and 75% of female Lmna^L648R/L648R mice having near-normal lifespans of 90 wk (almost 2 y). Starting at ∼10 wk of age, Lmna^L648R/L648R mice of both sexes have lower body masses than controls. By ∼20 to 30 wk of age, they exhibit detectable cranial, mandibular, and dental defects similar to those observed in Zmpste24^−/− mice and have decreased vertebral bone density compared to age- and sex-matched controls. Cultured embryonic fibroblasts from Lmna^L648R/L648R mice have aberrant nuclear morphology that is reversible by treatment with a protein farnesyltransferase inhibitor. These novel mice provide a model to study the effects of farnesylated prelamin A during physiological aging.

The lamin A/C gene (LMNA) encodes the splice variants lamin A and lamin C, which are intermediate filament building blocks of the nuclear lamina that differ in their carboxyl-terminal domains. Prelamin A, but not lamin C, has a carboxyl-terminal cysteine-aliphatic-aliphatic-any amino acid (CAAX) motif that initiates a series of posttranslational processing reactions to generate mature lamin A. In addition to CAAX processing (farnesylation, cleavage of -AAX, and carboxymethylation), prelamin A undergoes a final cleavage reaction, uniquely catalyzed by the zinc metalloprotease ZMPSTE24. This removes the last 15 amino acids of prelamin A, including its farnesylated cysteine, resulting in the production of mature, unfarnesylated lamin A. Defects in the posttranslational processing of prelamin to lamin A cause progeroid disorders (Fig. 1A) (1–3).Open in a separate windowFig. 1.Survival of mice with a Lmna L648R mutation corresponding to the human mutation LMNA L647R that encodes an uncleavable variant of prelamin A. (A) Prelamin A is normally processed to lamin A after proteolytic cleavage catalyzed by the zinc metalloprotease ZMPSTE24 between tyrosine (Y) and leucine (L) 647 (648 in mouse), removing a peptide containing the carboxyl-terminal farnesylated cysteine (C). HGPS-causing LMNA mutations generate a prelamin A variant with an internal deletion of 50 amino acids (Δ50) called progerin, which lacks the ZMPSTE24 cleavage site and retains a farnesylated carboxyl-terminal cysteine. ZMPSTE24 loss-of-function mutations cause RD or MAD-B, in which unprocessed farnesylated prelamin A accumulates. LMNA mutation causing a MAD-B-like disorder generates a leucine (L) to arginine (R) substitution at residue 647 of prelamin A blocks ZMPSTE24 processing, leading to expression of a farnesylated variant with only a single amino acid difference. (B) Immunoblots of protein extracts from livers of Lmna^+/+ (+/+), Lmna^+/L648R (+/L648R), and Lmna^L648R/L648R (L648R/L648R) mice. Blots were probed with an antibody specific for prelamin A (Top), an anti-lamin A/C antibody that recognized prelamin A, lamin A, and lamin C (Middle), or anti-GAPDH antibody as loading control (Bottom). (C) Survival curves for male L648R/L648R (n = 19), +/L648R (n = 17), and +/+ (n = 10) mice and female L648R/L648R (n = 11), +/L648R (n = 15), and +/+ (n = 8) mice.Hutchinson–Gilford progeria syndrome (HGPS) results from a splicing mutation in LMNA that generates a variant with a 50-amino acid internal deletion (Δ50) called progerin, which retains its CAAX motif but lacks the ZMPSTE24 cleavage site, and thus progerin’s carboxyl-terminal cysteine remains permanently farnesylated (4, 5). Children with HGPS manifest numerous premature aging symptoms, including failure to thrive, bone loss, and early-onset severe atherosclerosis leading to death typically in the second decade. Progeroid disorders also result from mutations in ZMPSTE24 that lead to accumulation of full-length farnesylated prelamin A. Mandibuloacral dysplasia type B (MAD-B) is generally less severe than HGPS and caused by partial loss-of-function mutations in ZMPSTE24, with disease severity correlating with residual proteolytic activity (6, 7). Restrictive dermopathy (RD), caused by complete loss-of-function mutations in ZMPSTE24, is neonatal lethal (8).Genetically modified mouse models provide valuable tools to study these human progeroid disorders and may have the potential to illuminate the role of permanently farnesylated prelamin A in physiologic aging. Zmpste24^−/− mice accumulate farnesylated prelamin A and develop progeroid phenotypes, including severe growth retardation, craniofacial abnormalities, spontaneous bone fractures, and have a median survival of only ∼20 wk (9, 10). In Zmpste24^−/− mice, disease severity is ameliorated by genetic reduction of prelamin A or pharmacological treatment to block protein farnesylation (11–13). These findings, along with those showing a correlation between disease severity and residual enzyme activity in humans with ZMPSTE24 mutations, suggest a “dose–response” between the amount of farnesylated prelamin A and the degree of pathology. However, ZMPSTE24 has at least one other function besides prelamin A processing, namely clearing clogged translocons (14, 15). Hence, some of the organismal pathology caused by ZMPSTE24 deficiency may result from defects in protein translocation into the endoplasmic reticulum (ER). ZMPSTE24 may also have additional functions, inferred from its contribution to viral defense in mice (16, 17) and from genetic studies in yeast that have implicated it in ER protein quality control, membrane stress, and establishing membrane protein topology (18–22).Substitution of a hydrophobic residue with an arginine at the ZMPSTE24 cleavage site in prelamin A (V637R in chickens and L647R in humans) blocks its proteolysis (23, 24), resulting in the accumulation of permanently farnesylated prelamin A. We have previously reported a patient with a heterozygous LMNA mutation generating the L647R amino acid substitution in prelamin A. This patient had a relatively mild progeroid disorder with clinical features similar to those of MAD-B (25). We therefore generated and characterized mice with the corresponding L648R amino acid substitution in prelamin A to determine if Lmna^L648R/L648R mice have the same or different phenotype and disease severity as Zmpste24^−/− mice.     

Autosomal dominant dilated cardiomyopathy with atrioventricular block: a lamin A/C defect-related disease

OBJECTIVES: We investigated the prevalence of lamin A/C (LMNA) gene defects in familial and sporadic dilated cardiomyopathies (DCM) associated with atrioventricular block (AVB) or increased serum creatine-phosphokinase (sCPK), and the corresponding changes in myocardial and protein expression. BACKGROUND: It has been reported that familial DCM, associated with conduction disturbances or variable myopathies, is causally linked to LMNA gene defects. METHODS: The LMNA gene and myocardial ultrastructural and immunochemical changes were analyzed in 73 cases of DCM (49 pure, 15 with AVB [seven familial, eight sporadic], 9 with increased sCPK), four cases of familial AVB and 19 non-DCM heart diseases. The normal controls included eight heart donor biopsies for tissue studies and 107 subjects for LMNA gene studies. RESULTS: Five novel LMNA mutations (K97E, E111X, R190W, E317K, four base pair insertion at 1,713 cDNA) were identified in five cases of familial autosomal dominant DCM with AVB (5/15: 33%). The LMNA expression of the myocyte nuclei was reduced or absent. Western blot protein analyses of three hearts with different mutations showed an additional 30-kDa band, suggesting a degrading effect of mutated on wild-type protein. Focal disruptions, bleb formation and nuclear pore clustering were documented by electron microscopy of the myocyte nuclear membranes. None of these changes and no mutations were found in the nine patients with DCM and increased sCPK or in the disease and normal controls. CONCLUSIONS: The LMNA gene mutations account for 33% of the DCMs with AVB, all familial autosomal dominant. Increased sCPK in patients with DCM without AVB is not a useful predictor of LMNA mutation.     

Phenotypic heterogeneity in patients with familial partial lipodystrophy (dunnigan variety) related to the site of missense mutations in lamin a/c gene

The lamin A/C (LMNA) gene has recently been reported to be mutated in familial partial lipodystrophy, Dunnigan variety (FPLD). We found mutations within exon 8 of LMNA (R482Q, R482W, and G465D) in 12 families with typical FPLD and in exon 11 (R582H) in 1 atypical family. To investigate phenotypic heterogeneity, we compared body fat distribution, using anthropometry and whole body magnetic resonance imaging, and metabolic parameters in women with atypical and typical FPLD. Compared with women with typical FPLD, the two sisters with atypical FPLD had less severe loss of sc fat from all the extremities and trunk and particularly from the gluteal region and medial parts of proximal thighs. Both types had similar excess of fat deposition in the neck, face, intraabdominal, and intermuscular regions. Women with atypical FPLD tended to have lower serum triglyceride and higher high density lipoprotein cholesterol concentrations. As exon 11 of LMNA does not comprise part of the lamin C-coding region, the R582H mutation affects only lamin A protein. Therefore, a unique phenotype of atypical FPLD may result from disrupted interaction of lamin A with other proteins and chromatin compared with typical FPLD, in which interaction of both lamins A and C may be disrupted.     

Body fat distribution in women with familial partial lipodystrophy caused by mutation in the lamin A/C gene

Familial partial lipodystrophy (FPLD), Dunnigan variety, is an autosomal dominant disorder caused due to missense mutations in the lamin A/C (LMNA) gene encoding nuclear lamina proteins. Patients with FPLD are predisposed to metabolic complications of insulin resistance such as diabetes. We sought to evaluate and compare body fat distribution with dual-emission X-ray absorptiometry in women with and without FPLD and identify densitometric, clinical and metabolic features.