Association between nuclear lamin A/C R482Q mutation and partial lipodystrophy with hyperinsulinemia, dyslipidemia, hypertension, and diabetes

Nuclear lamins A and C are encoded by LMNA and are present in terminally differentiated cells. Lamins participate in DNA replication, chromatin organization, arrangement of nuclear pores, nuclear growth, and anchorage of nuclear membranes. In several Canadian probands with partial lipodystrophy, since found to have a common ancestor, we identified a rare novel LMNA mutation, R482Q, that completely cosegregated with the partial lipodystrophy phenotype. We evaluated the relationship between quantitative metabolic phenotypes in both diabetic and nondiabetic carriers of LMNA R482Q and family controls, who were LMNA R482/R482 homozygotes. We found that when compared with LMNA R482/R482 homozygotes: (1) diabetic LMNA Q482/R482 heterozygotes had significantly higher glucose, glycosylated hemoglobin, triglycerides, insulin and C-peptide, and significantly lower HDL cholesterol; and (2) nondiabetic LMNA Q482/R482 heterozygotes had significantly higher triglycerides, insulin and C-peptide, and significantly lower HDL cholesterol. We also found that diabetic LMNA Q482/R482 heterozygotes were older and more likely to take antihypertensive medications. Thus, LMNA R482Q was associated with lipodystrophy, hyperinsulinemia, dyslipidemia, diabetes, and hypertension. The results indicate that perturbations in plasma lipids precede the plasma glucose abnormalities in LMNA Q482-associated hyperinsulinemia. Thus, rare mutations in a nuclear structural protein can be associated with markedly abnormal qualitative and quantitative metabolic phenotypes     

Nuclear lamin A/C R482Q mutation in canadian kindreds with Dunnigan-type familial partial lipodystrophy

Patients with Dunnigan-type familial partial lipodystrophy (FPLD) are born with normal fat distribution, but after puberty experience regional and progressive adipocyte degeneration, often associated with profound insulin resistance and diabetes. Recently, the FPLD gene was mapped to chromosome 1q21-22, which harbours the LMNA gene encoding nuclear lamins A and C. Mutations in LMNA were shown to underlie autosomal dominant Emery-Dreifuss muscular dystrophy (EDMD-AD), which is characterized by regional and progressive skeletal muscle wasting and cardiac effects. We hypothesized that the analogy between the regional muscle wasting in EDMD-AD and the regional adipocyte degeneration in FPLD, in addition to its chromosomal localization, made LMNA a good candidate gene for FPLD. DNA sequencing of LMNA in five Canadian FPLD probands indicated that each had a novel missense mutation, R482Q, which co-segregated with the FPLD phenotype and was absent from 2000 normal alleles ( P = 1.1 x 10(-13)). This is the first report of a mutation underlying a degenerative disorder of adipose tissue and suggests that LMNA mutations could underlie other diseases characterized by tissue type- and anatomical site-specific cellular degeneration.     

LMNA突变体HEK293、C2C12模型的构建及其核纤层蛋白A/C亚细胞定位

目的构建A型核纤层蛋白基因(LMNA)野生型、突变体的融合蛋白真核表达载体及LMNA突变体慢病毒载体,研究其在HEK293、C2C12中表达、核纤层蛋白A/C亚细胞定位及细胞核改变。方法将野生型全长LMNA cDNA克隆入pEGFP-N1质粒,构建LMNA野生型质粒pEGFP-N1-LMNA,以野生型质粒为模板构建c.1117A>G定点突变质粒pEGFP-N1-LMNA-I373V。将构建的2种质粒分别转染HEK293、C2C12,用G418筛选转染的C2C12,荧光显微镜下观察细胞核形态及GFP标记的核纤层蛋白A/C亚细胞定位。以pEGFP-N1-LMNA-I373V为模板,构建pHBLV-h-LMNA-I373V-3*flag-GFP-PURO慢病毒,对慢病毒进行包装与滴度测定。pHBLV-GFP-PURO、pHBLV-LMNA-C1117-3*flag-GFP-PURO分别转染C2C12,免疫荧光染色法观察转染后细胞核形态及核纤层蛋白A/C亚细胞定位的改变。结果构建的pEGFP-N1-LMNA、pEGFP-N1-LMNA-I373V及pHBLV-h-LMNA-I373V-3*flag-GFP-PURO测序与目的基因序列完全一致。pEGFP-N1-LMNA转染的HEK293、C2C12核纤层蛋白A/C均匀表达于核膜下,pEGFP-N1-LMNA-I373V转染的HEK293、C2C12内核纤层蛋白A/C核内异常聚集,呈散点样分布;与HEK293相比,C2C12细胞转染效率明显降低。慢病毒转染C2C12的转染率高,突变体慢病毒转染的细胞核形态异常及核纤层蛋白A/C核内分布异常。结论成功构建2种LMNA突变体真核表达载体及突变体转染的HEK293、C2C12模型,为LMNA突变体导致疾病的机制研究奠定科学基础。     

The MuLV 4070A G541R Env mutation decreases the stability and alters the conformation of the TM ectodomain

Virus-cell and cell-cell fusion events are affected by various properties of the fusogenic Env protein on the cell surface. The G541R mutation within the TM ectodomain of murine leukemia virus (MuLV) 4070A arose by positive selection in viral passage and results in a reduction of cell-cell fusion events while maintaining viral titer. Size exclusion chromatography shows that the multimerization properties are similar among expressed wild-type and mutant ectodomain peptides. Circular dichroism measurements reveal decreased thermal stability of the G541R mutant as compared to wild type. The G541R mutant also renders the peptide more susceptible to Lys-C protease cleavage. The 42-114 monoclonal antibody does not bind to the G541R mutant peptides, suggesting a structural difference from wild type. These altered physical properties result in productive viral infection of G541R bearing virus with decreased syncytia.     

Association of homozygous LMNA mutation R471C with new phenotype: mandibuloacral dysplasia, progeria, and rigid spine muscular dystrophy

We report on a 7-year-old girl with a phenotype combining mandibuloacral dysplasia (MAD), progeria, and rigid spine muscular dystrophy. Mild proximal weakness, contractures, and rigidity of the spine were the primary findings. Although present since birth, dysmorphic manifestations typical for MAD and progeroid features became more prominent with time, and the full clinical phenotype was recognizable at early school age. Her phenotype was caused by a homozygous mutation in LMNA (c.1411C > T, which predicts p.R471C) inherited from the heterozygous, consanguineous, unaffected parents. This mutation has only been reported in compound heterozygous state and was associated with a milder phenotype. Some LMNA mutations are known to cause MAD and overlapping phenotypes (MAD spectrum) in an autosomal recessive pattern. The p.R471C homozygous LMNA mutation causes a severe phenotype of the MAD spectrum. This case extends the clinical spectrum of MAD and further expands the phenotypic range of lamin A/C associated diseases.     

Mutations in the LMNA gene encoding lamin A/C

Very recently, mutations within the LMNA gene on chromosome 1q21.2 were shown to result in forms of muscular dystrophy, conduction-system disease, cardiomyopathy, and partial lipodystrophy. The LMNA gene encodes for the nucleophilic A-type lamins, lamin A and lamin C. These isoforms are generated by different splicing within exon 10 of LMNA. Thus lamin A/C is, besides emerin, the first known nucleophilic protein which plays a role in human disease. To date, 41 different mutations, predominantly missense, in the LMNA gene are known causing variable phenotypes. Twenty-three different mutations of LMNA have so far been shown to cause autosomal-dominant Emery-Dreifuss muscular dystrophy (EDMD2), three mutations were reported to cause limb-girdle muscular dystrophy (LGMD1B), eight mutations are known to result in dilated cardiomyopathy (CMD1A), and seven mutations were reported to cause familial partial lipodystrophy (FPL). The reports of lamin mutations including the corresponding phenotype are of great interest in order to gain insights into the function of lamin A/C. Here we summarize the mutations published to date in LMNA encoding lamin A/C.     

Clinical and mutational spectrum in a cohort of 105 unrelated patients with dilated cardiomyopathy

Dilated Cardiomyopathy (DCM) is one of the leading causes of heart failure with high morbidity and mortality. More than 30 genes have been reported to cause DCM. To provide new insights into the pathophysiology of dilated cardiomyopathy, a mutational screening on 4 DCM-causing genes (MYH7, TNNT2, TNNI3 and LMNA) was performed in a cohort of 105 unrelated DCM (64 familial cases and 41 sporadic cases) using a High Resolution Melting (HRM)/sequencing strategy. Screening of a highly conserved arginine/serine (RS)-rich region in exon 9 of RBM20 was also performed. Nineteen different mutations were identified in 20 index patients (19%), including 10 novels. These included 8 LMNA variants in 9 (8.6%) probands, 5 TNNT2 variants in 5 probands (4.8%), 4 MYH7 variants in 3 probands (3.8%), 1 TNNI3 variant in 1 proband (0.9%), and 1 RBM20 variant in 1 proband (0.9%). One proband was double-heterozygous. LMNA mutations represent the most prevalent genetic DCM cause. Most patients carrying LMNA mutations exhibit conduction system defects and/or cardiac arrhythmias. Our study also showed than prevalence of mutations affecting TNNI3 or the (RS)-rich region of RBM20 is lower than 1%. The discovery of novel DCM mutations is crucial for clinical management of patients and their families because pre-symptomatic diagnosis is possible and precocious intervention could prevent or ameliorate the prognosis.     

Sensitive multistep clinical molecular screening of 180 unrelated individuals with retinoblastoma detects 36 novel mutations in the RB1 gene

Thymopoietin or TMPO (indicated by its alternative gene symbol, LAP2, in this work) has been proposed as a candidate disease gene for dilated cardiomyopathy (DCM), since a LAP2 product associates with nucleoplasmic lamins A/C, which are encoded by the DCM gene LMNA. We developed a study to screen for genetic mutations in LAP2 in a large collection of DCM patients and families. A total of 113 subjects from 88 families (56 with familial DCM (FDC) and 32 with sporadic DCM) were screened for LAP2 mutations using denaturing high-performance liquid chromatography and sequence analysis. We found a single putative mutation affecting the LAP2alpha isoform in one FDC pedigree. The mutation predicts an Arg690Cys substitution (c.2068C>T; p.R690C) located in the C-terminal domain of the LAP2alpha protein, a region that is known to interact with lamin A/C. RT-PCR, Western blot analyses, and immunolocalization revealed low-level LAP2alpha expression in adult cardiac muscle, and localization to a subset of nuclei. Mutated Arg690Cys LAP2alpha expressed in HeLa cells localized to the nucleoplasm like wild-type LAP2alpha, with no effect on peripheral and nucleoplasmic lamin A distribution. However, the in vitro interaction of mutated LAP2alpha with the pre-lamin A C-terminus was significantly compromised compared to the wild-type protein. LAP2 mutations may represent a rare cause of DCM. The Arg690Cys mutation altered the observed LAP2alpha interaction with A-type lamins. Our finding implicates a novel nuclear lamina-associated protein in the pathogenesis of genetic forms of dilated cardiomyopathy.     

Doubly heterozygous LMNA and TTN mutations revealed by exome sequencing in a severe form of dilated cardiomyopathy

Familial dilated cardiomyopathy (DCM) is a heterogeneous disease; although 30 disease genes have been discovered, they explain only no more than half of all cases; in addition, the causes of intra-familial variability in DCM have remained largely unknown. In this study, we exploited the use of whole-exome sequencing (WES) to investigate the causes of clinical variability in an extended family with 14 affected subjects, four of whom showed particular severe manifestations of cardiomyopathy requiring heart transplantation in early adulthood. This analysis, followed by confirmative conventional sequencing, identified the mutation p.K219T in the lamin A/C gene in all 14 affected patients. An additional variant in the gene for titin, p.L4855F, was identified in the severely affected patients. The age for heart transplantation was substantially less for LMNA:p.K219T/TTN:p.L4855F double heterozygotes than that for LMNA:p.K219T single heterozygotes. Myocardial specimens of doubly heterozygote individuals showed increased nuclear length, sarcomeric disorganization, and myonuclear clustering compared with samples from single heterozygotes. In conclusion, our results show that WES can be used for the identification of causal and modifier variants in families with variable manifestations of DCM. In addition, they not only indicate that LMNA and TTN mutational status may be useful in this family for risk stratification in individuals at risk for DCM but also suggest titin as a modifier for DCM.     

Cells expressing partially unfolded R789C/p.R989C type II procollagen mutant associated with spondyloepiphyseal dysplasia undergo apoptosis

We investigated the effects of the presence of R75C (p.R275C), R519C (p.719C), R789C (p.R989C), and G853E (p.G1053E) type II collagen (COL2A1) mutants, associated with distinct forms of spondyloepiphyseal dysplasia (SED), on the biological processes occurring in chondrocytic cells harboring those mutants. Mutant-specific biological responses of cells were initiated by activating tetracycline (Tet)-dependent expression of type II collagen mutants. Employing microscopic and biochemical assays, we determined that cells expressing the thermolabile R789C (p.R989C) type II collagen mutant undergo apoptosis. In contrast, in cells expressing the thermostable R75C (p.R275C), R519C (p.719C), and G853E (p.G1053E) mutants, apoptotic markers were not apparent. We also demonstrated that the R789C (p.R989C) mutant formed atypical complexes with endoplasmic reticulum (ER)-resident chaperones, thereby indicating an "unfolded protein response" (UPR) of cells harboring this specific mutant. Apoptotic changes were also demonstrated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and cleaved caspase 3 assays in the growth plates of mice harboring the R992C (p.R1147C) substitution in type II collagen. Based on these results, we propose that the intracellular presence of structurally altered type II collagen mutants could activate an apoptotic response, thereby limiting cell survival. By analyzing the response of cells to the altered structure of collagen mutants, our study contributes to better understanding the molecular basis of the pathological changes seen in vivo at the tissue level.     

The heterozygous LMNA mutation p.R471G causes a variable phenotype with features of two types of familial partial lipodystrophy

We report on a novel LMNA mutation (p.R471G) in a proband affected by a syndrome comprising partial lipodystrophy, insulin-resistant diabetes, acanthosis nigricans, liver steatosis, muscle weakness, and contractures. This phenotype has features of both types 1 and 2 familial partial lipodystrophy. The sister and father of the proband had the same mutation. The sister was more mildly affected and the father was apparently unaffected, demonstrating variable expressivity and reduced penetrance for this mutation.     

Novel and recurrent mutations in lamin A/C in patients with Emery-Dreifuss muscular dystrophy

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by slowly progressive muscle wasting and weakness; early contractures of the elbows, Achilles tendons, and spine; and cardiomyopathy associated with cardiac conduction defects. Clinically indistinguishable X-linked and autosomal forms of EDMD have been described. Mutations in the STA gene, encoding the nuclear envelope protein emerin, are responsible for X-linked EDMD, while mutations in the LMNA gene encoding lamins A and C by alternative splicing have been found in patients with autosomal dominant, autosomal recessive, and sporadic forms of EDMD. We report mutations in LMNA found in four familial and seven sporadic cases of EDMD, including seven novel mutations. Nine missense mutations and two small in-frame deletions were detected distributed throughout the gene. Most mutations (7/11) were detected within the LMNA exons encoding the central rod domain common to both lamins A/C. All of these missense mutations alter residues in the lamin A/C proteins conserved throughout evolution, implying an essential structural and/or functional role of these residues. One severely affected patient possesed two mutations, one specific to lamin A that may modify the phenotype of this patient. Mutations in LMNA were frequently identified among patients with sporadic and familial forms of EDMD. Further studies are needed to identify the factors modifying disease phenotype among patients harboring mutations within lamin A/C and to determine the effect of various mutations on lamin A/C structure and function.     

Laminopathies in Russian families

Mutations in LMNA gene produce a wide spectrum of disorders called laminopathies. In this article, the first cases of laminopathies from Russia are reported. In 10 unrelated families, 9 different mutations were identified: Asp47His, Gly232Arg, c.[781_783delAAG, 781insGTGGAGCAGTATAAGAAA], Arg249Gln (in two families), Arg377His, Arg541His, Ala350Pro, Leu52Pro, and Gly635Asp. Mutations Arg249Gln, Arg377His, and Arg541His were reported previously, others are novel. Four cases present de novo mutations, among them two cases with Arg249Gln are found. Because this mutation occurred de novo also in other reported cases, a mutational 'hot spot' was supposed. Three phenotypes were observed: autosomal dominant (AD) Emery–Dreifuss muscular dystrophy (EDMD), limb-girdle MD type 1B, and AD dilated cardiomyopathy with conduction defect type 1A (DCM1A). Atypical clinical presentations were a very severe EDMD and an infantile DCM1A.     

DNA spatial considerations in the arrangement of G/C and A/T blocks.

Intensive computations have been carried out on eukaryotic and prokaryotic DNA sequences present in the GenBank database. These calculations were aimed at studying particular sequence patterns. Previously we have observed a trend in the relative positioning of DNA oligomers with respect to each other. Specifically, we have shown that there is a preference for A/T stretches to be inserted inside G/C blocks, partitioning the latter. This arrangement is preferred over having a longer G/C block with an A/T stretch next to it. That is (G/C)n(A/T)m(G/C)2 greater than (G/C)n + 2(A/T)m. Previously we have attributed this preferred pattern to nucleosome packaging of the DNA. Since the average length of the DNA involved in a single nucleosome formation is 200 bp, oligomers were scored in the analysis only if two identical ones occurred within that distance. Since, in addition, the total length of the oligomers studied is n + m + 2 less than or equal to 7, the counts of the longer oligomers were quite low. Here we have repeated the analysis on the newer, larger database, with that restriction removed. Our new analysis confirms and strengthens the older findings. Moreover, we have carried out DNA structural analysis of these trends, focusing on the twist and roll angular parameters. We have obtained these values from detailed structural computations on the Cray super computer. Some correlations between these values, DNA flexibility and the sequence trends are observed. We suggest that the positioning of the (A/T)m sequence stretch between the two (G/C) blocks affords greater flexibility in the spatial positioning of the G/C sequence elements.     

Association of CD1A +622 T/C, +737 G/C and CD1E +6129 A/G genes polymorphisms with multiple sclerosis

Antibodies and specific T cells to glycolipids have been found in MS patients. CD1 molecules are involved in presentation of lipid antigens to T-cells. Therefore, functional polymorphisms in two CD1 genes (+622 T/C and +737 G/C in CD1A along with +6129 A/G in CD1E) might be associated with susceptibility to MS. First, 351 MS patients and 342 controls were enrolled in this study. Allele-specific oligonucleotide polymerase chain reaction and PCR-RFLP methods were used for genotyping. The frequency of CD1A genotypes was not different between cases and controls. However, investigating females, the frequency of CD1A*01 allele was significantly higher in patients with PP-MS compared to controls (p = 0.028) as well as to RR-MS and SP-MS (p = 0.042 and 0.021, respectively). The distribution of CD1E +6129 A allele (CD1E*01) and CD1E*01/01 genotype is more frequent in normal controls in comparison with MS patients (p = 0.001 and p = 0.0003, respectively). In addition, after categorization of study groups according to disease types, differences between alleles and genotypes of CD1E gene polymorphism remained significant for RR-MS patients compared to those of normal controls (p = 0.0001 and p = 0.0003, respectively). CD1E and CD1A genes may be involved in networks which determine susceptibility to RR-MS and PP-MS, respectively.     

Association of CD1A +622 T/C, +737 G/C and CD1E +6129 A/G Genes Polymorphisms with Multiple Sclerosis

Antibodies and specific T cells to glycolipids have been found in MS patients. CD1 molecules are involved in presentation of lipid antigens to T-cells. Therefore, functional polymorphisms in two CD1 genes (+622 T/C and +737 G/C in CD1A along with +6129 A/G in CD1E) might be associated with susceptibility to MS. First, 351 MS patients and 342 controls were enrolled in this study. Allele-specific oligonucleotide polymerase chain reaction and PCR-RFLP methods were used for genotyping. The frequency of CD1A genotypes was not different between cases and controls. However, investigating females, the frequency of CD1A*01 allele was significantly higher in patients with PP-MS compared to controls (p = 0.028) as well as to RR-MS and SP-MS (p = 0.042 and 0.021, respectively). The distribution of CD1E +6129 A allele (CD1E*01) and CD1E*01/01 genotype is more frequent in normal controls in comparison with MS patients (p = 0.001 and p = 0.0003, respectively). In addition, after categorization of study groups according to disease types, differences between alleles and genotypes of CD1E gene polymorphism remained significant for RR-MS patients compared to those of normal controls (p = 0.0001 and p = 0.0003, respectively). CD1E and CD1A genes may be involved in networks which determine susceptibility to RR-MS and PP-MS, respectively.     

PLA2G6 variant in Parkinson's disease

PLA2G6 was reported recently as the causative gene for PARK14-linked autosomal recessive early-onset dystonia-parkinsonism. In a recent study in Singapore, heterozygous PLA2G6 p.P806R (c.2417C>G) mutation in exon 17 was reported to be a possible Parkinson's disease (PD)-related mutation. To determine the significance of the PLA2G6 mutation, we conducted an association study by performing direct sequencing of PLA2G6 exon 17 in 379 Japanese sporadic PD patients and 310 controls in the Japanese general population. In this group, we found 12 patients (12/379=3.16%) and 10 controls (10/310=3.23%) with a heterozygous p.P806R mutation (P=0.96, χ(2)=0.0019). Therefore, our large case-controlled study suggests that PLA2G6 p.P806R is not a disease-associated polymorphism in PD. Moreover, we performed direct sequencing of all exons and exon-intron boundaries of PLA2G6 in 116 Japanese patients with sporadic PD. Two single heterozygous variants (p.R301C or p.D331N) were found (both frequencies: 1/379 patients vs 0/310 controls) and the roles of their variants were unclear. Finally, combined with the previous report, our findings emphasize that PLA2G6 mutations are unlikely to be the major causes or risk factors of PD at least in Asian populations. However, further large studies in various populations are needed because patients with PLA2G6 mutations can show heterogeneous clinical features.