Author + information
- Received April 9, 2012
- Revision received May 23, 2012
- Accepted May 28, 2012
- Published online November 6, 2012.
- Nupoor Narula, BS⁎,
- Valentina Favalli, BME⁎,
- Paolo Tarantino, BD⁎,
- Maurizia Grasso, PhD⁎,
- Andrea Pilotto, BS⁎,
- Riccardo Bellazzi, PhD†,
- Alessandra Serio, MD⁎,
- Fabiana I. Gambarin, MD⁎,
- Philippe Charron, MD‡,
- Benjamin Meder, MD§,
- Yigal Pinto, MD∥,
- Perry M. Elliott, MD¶,
- Jens Mogensen, MD#,
- Martino Bolognesi, PhD⁎⁎,
- Michela Bollati, PhD⁎⁎ and
- Eloisa Arbustini, MD⁎,⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Eloisa Arbustini, Centre for Inherited Cardiovascular Diseases, IRCCS Fondazione Policlinico San Matteo, Piazzale Golgi 19, 27100 Pavia, Italy
Objectives The authors sought to investigate the gene and protein expression in Lamin A/C (LMNA)-mutated dilated cardiolaminopathy (DCM) patients (DCMLMNAMut) versus LMNA-wild-type DCM (DCMLMNAWT), and normal controls (CTRLLMNAWT).
Background Dilated cardiolaminopathies are clinically characterized by high arrhythmogenic risk and caused by LMNA mutations. Little is known regarding quantitative gene expression (QGE) of the LMNA gene in blood and myocardium, as well as regarding myocardial expression of the lamin A/C protein.
Methods Using the comparative ΔΔCT method, we evaluated the QGE of LMNA (QGELMNA) in peripheral blood and myocardial RNA from carriers of LMNA mutations, versus blood and myocardial samples from DCMLMNAWT patients and CTRLLMNAWT individuals. After generating reference values in normal controls, QGELMNA was performed in 311 consecutive patients and relatives, blind to genotype, to assess the predictive value of QGELMNA for the identification of mutation carriers. In parallel, Lamin A/C was investigated in myocardial samples from DCMLMNAMut versus DCMLMNAWT versus normal hearts (CTRLLMNAWT).
Results LMNA was significantly underexpressed in mRNA from peripheral blood and myocardium of DCMLMNAMut patients versus DCMLMNAWT and CTRLLMNAWT. In 311 individuals, blind to genotype, the QGELMNA showed 100% sensitivity and 87% specificity as a predictor of LMNA mutations. The receiver-operating characteristic curve analysis yielded an area under the curve of 0.957 (p < 0.001). Loss of protein in cardiomyocytes' nuclei was documented in DCMLMNAMut patients.
Conclusions The reduced expression of LMNA gene in blood is a novel potential predictive biomarker for dilated cardiolaminopathies with parallel loss of protein expression in cardiomyocyte nuclei.
Dilated cardiomyopathy (DCM) is a primary myocardial disease with heterogeneous etiology, wherein genetic defects are found in up to 48% of DCM cases (1). Lamin A/C (LMNA) mutations occur in approximately 8% of familial cases (2); the most common cardiac phenotype associated with LMNA defects comprises DCM with conduction disease (80%), high arrhythmogenic risk, and progression to end-stage heart failure (3–5). Haploinsufficiency is a major mechanism of myocardial damage in Lmna+/− mouse models (6). In human laminopathy, the expression of the protein in the nuclear membrane of cardiac and skeletal myocytes may either be reduced or remain normal (4,7). Although quantitative gene expression (QGE) in peripheral blood has been suggested as a promising molecular signature of DCM (8), only scattered data exist of the mutated LMNA gene (QGELMNA) in patients with cardiolaminopathy (9).
To investigate the expression of lamin A/C protein and QGELMNA as well as its potential diagnostic contribution to the clinical work-up of cardiomyopathy, we determined immunohistochemical expression of the lamin A/C protein in the myocardium and QGELMNA in RNA from myocardial tissue and peripheral blood samples of DCM patients with LMNA mutations.
The study was designed to measure LMNA gene expression in DCM patients with known LMNA mutations (DCMLMNAMut) in comparison with both DCM patients with wild-type LMNA (DCMLMNAWT) and healthy control subjects (CTRLLMNAWT) (Online Fig. 1). Methods used for genetic testing of LMNA and for QGELMNA at both the myocardial and the peripheral blood levels are detailed in the Online Methods.
In the first part of the study, we established the normal QGELMNA values in 115 CTRLLMNAWT subjects for the evaluation of 96 DCMLMNAWT and 67 DCMLMNAMUT patients. In the second part, we aimed to assess the value of the QGELMNA assay for predicting LMNA mutations in 311 individuals, both DCM probands and relatives, blinded to genotype. Online Table 1 summarizes the mutations, the number of patients that were included in the first study, and the number of mutated patients and healthy carriers that were included in the second study. In the third part, directed at myocardial tissue, we also performed QGELMNA in endomyocardial biopsy (EMB) samples obtained from 25 DCMLMNAMUT prospective heart transplant recipients and compared them with 20 CTRLLMNAWT endomyocardial biopsies obtained from donor hearts before transplantation. In addition to QGELMNA, using anti-lamin A/C antibodies (4), the protein expression was determined in 25 myocardial samples of patients with DCMLMNAMut, 20 DCMLMNAWT, and 20 CTRLLMNAWT. Western blot analysis was undertaken in 12 DCMLMNAMut and 9 control heart samples with wild-type LMNA (Online Methods). We managed to collect appropriate samples for RNA in 22 patients with known laminopathy on 2 different occasions to assess whether the QGELMNA would change during follow-up. The study complies with the Declaration of Helsinki and was approved by the local ethics committee. All study participants provided written informed consent for genetic testing and anonymous data publication for scientific purposes.
For QGELMNA, relative quantification of the LMNA RNA at each time point was determined using the comparative ΔΔCT method (10) (Online Materials). The Kruskal-Wallis nonparametric test was used for multiple comparisons, using Mann-Whitney rank sum test with post hoc Bonferroni correction. The Mann-Whitney rank sum test was used for nonmultiple comparisons. A p value <0.05 was considered significant. Stata 10.1 (StataCorp, College Station, Texas) was used for computation. The receiver-operating characteristic (ROC) curve analysis (11) was performed in the 311 cases blind to mutations.
Peripheral blood study: LMNA mRNA levels in known laminopathy and controls
Expression of LMNA mRNA Levels in Patients With Known DCMLMNAMut
The QGELMNA referral value in CTRLLMNAWT individuals was 1.92. LMNA was underexpressed by 30% in DCMLMNAMut patients as compared with CTRLLMNAWT individuals (2−ΔCt ± SEM = 1.32 ± 0.10 vs. 1.92 ± 0.18, respectively; p < 0.007) (Fig. 1A), and by 26% (2−ΔCt ± SEM = 1.32 ± 0.10 vs. 1.76 ± 0.16; p = 0.02) as compared with DCMLMNAWT patients. Values observed in DCMLMNAWT versus CTRLLMNAWT were not significantly different (p = 0.71).
LMNA mRNA Levels and Mutation Type
Comparative evaluation of LMNA mRNA levels among groups of patients with different types of LMNA mutations (missense, n = 37; in-frame insertion/deletion, n = 7; nonsense mutations and frameshift insertions/deletions, predicting a premature termination codon [PTC] and truncated lamin A/C protein [n = 12]; and splice site [n = 11]) did not demonstrate significant differences (p = 0.93) (Fig. 1B).
LMNA mRNA Levels and Mutation Position
QGELMNA did not differ in carriers of mutations up- and downstream of the nuclear localizing sequence (NLS) (2−ΔCt ± SEM = 1.35 ± 0.10 and 1.18 ± 0.33; p = 0.75), all showing significantly lower levels when compared with controls (upstream NLS 2−ΔCt ± SEM = 1.35 ± 0.10 vs. CTRLLMNAWT = 1.92 ± 0.18; p = 0.01) and 39% (downstream NLS 2−ΔCt ± SEM = 1.18 ± 0.33 vs. CTRLLMNAWT = 1.92 ± 0.18; p = 0.007), respectively (Fig. 1C).
Peripheral blood study: LMNA mRNA levels in 311 consecutive patients and relatives, blinded to genotype
The ROC analysis (Fig. 1D) yielded an area under the curve of 0.957 (95% confidence interval: 0.928 to 0.977) with significance level p < 0.001, confirming the QGELMNA value as a robust possible predictor of mutation presence. On the basis of the sensitivity and specificity of the assay, we tested 3 possible ratios as threshold values: 0.60, 0.70, and 0.75 (Table 1). Since our major aim was to predict LMNA mutations, we selected the threshold value of 0.75 that showed the highest sensitivity (100%) but the lowest specificity (87%). Using this threshold, the QGELMNA assay predicted not only all mutation carriers, but also 30 patients/individuals with the wild-type LMNA gene. (Online Table 2 lists QGELMNA data in the 311 samples, blind to genotype before QGELMNA.)
In the 22 LMNA mutation carriers with at least 2 samples obtained at different intervals (2.18 ± 1.43 years), LMNA gene expression showed a trend to decrease with time (Online Table 3). A possible future relevance of comparative evaluation over time is related to the potential use of QGELMNA for monitoring the evolution of the disease or correlation with functional data or medical treatment.
Myocardial tissue studies
EMB of DCMLMNAMut patients revealed prominent nuclear changes, including irregularities of the morphological profile of myocyte nuclei and increased chromatin density, multifocal myofibrillar loss, and fibrosis of variable extent and severity. Ultrastructural study demonstrated severe changes in the nuclear membranes with blebs, pore clustering, nuclear microtubular structures, and pseudoinclusions (Fig. 2G).
LAMIN A/C Immunostain and Western Blot
The EMBs of CTRLLMNAWT and of DCMLMNAWT patients demonstrated homogeneous protein localization throughout the nuclear membrane of the nuclei of myocytes (Figs. 2A and 2B). Myocytes of DCMLMNAMut patients exhibited variable loss of lamin A/C expression (Figs. 2C to 2F), whereas interstitial and endothelial cells showed normal expression of the protein. Decreased protein expression was confirmed by Western blot analysis (Fig. 2H).
LMNA Gene Expression in Myocardial Tissue
There was 71% underexpression of LMNA in the myocardial samples of DCMLMNAMut patients (2−ΔCt ± SEM = 41.56 ± 12.06) as compared with myocardium from CTRLLMNAWT (2−ΔCt ± SEM = 143.72 ± 2.59) with a ratio of mutated versus wild type of 29% (p < 0.001) (Fig. 1E).
The present study documents that DCMLMNAMut patients and healthy mutation carriers show decreased expression of the mutated gene in the mRNA in peripheral blood samples as well as the affected myocardium when compared with both normal controls and DCM patients with wild-type LMNA. In addition, the LMNA protein is underexpressed at the nuclear level in the cardiac myocytes of cardiolaminopathy patients.
The QGELMNA assay is a potential biomarker for cardio-laminopathies
LMNA gene expression decreases independently of mutation type in myocardial RNA. Although to a lower extent, a similar decrease is observed in the corresponding peripheral blood mRNA. Given the lower cost and the ease of performing the QGE compared with the genetic test, it could constitute a pre-genetic assay for guiding LMNA gene testing, which is the diagnostic gold standard. However, whereas the QGELMNA assay for the threshold ratio value of 0.75 recognized all mutated patients, it also showed a false-positive result in 30 patients with the wild-type LMNA gene.
The decreased gene and protein expression confirms haplo-insufficiency as major disease mechanism in humans
The loss of lamin A/C in cardiac myocytes in vivo supports the haploinsufficiency mechanism previously shown in experimental models (6). Actively cycling cell types, including endothelial and interstitial cells, displayed normal LMNA immunostaining as compared with the gradual decrease in type-A lamins in the terminally differentiated cardiac myocytes. Low LMNA mRNA levels in the terminally differentiated, quiescent cells preclude the possibility of increased LMNA mRNA translation to stabilize the degradation rate, whereas the increased mRNA levels in cycling cells moderately compensates for the progressive loss of protein (12).
In carriers of splice site and PTC mutations, the low levels of LMNA mRNA are likely explained by the rapid degradation of transcripts with PTC mutations by nonsense mediated decay as suggested in a family with dilated cardiolaminopathy harboring the p.Arg321X LMNA mutation (13). In carriers of missense LMNA mutations, the low levels of LMNA mRNA are likely due to modifications of the position and compaction of chromatin (14). The tethering of mammalian genes to the nuclear lamina leads to transcriptional repression possibly as a result of separation from the RNA Polymerase II machinery or formation of a repressive chromatin structure (9). The key role of lamins in the structure and function of nuclei suggests that gene-specific and genome-wide chromatin rearrangements may contribute to molecular mechanisms of pathology observed in patients with LMNA mutations (15).
Decreased expression of Lamin A/C in myocardial samples
Lamin A/C protein expression is decreased in myocyte nuclei of affected hearts, independently of mutation type. The underexpression of the protein in vivo has been previously shown in the myocardium of patients with dilated cardiolaminopathy (4), and is now confirmed, thus being a diagnostic contributor if EMB is performed. The decreased amount of lamin A/C is also confirmed by Western blot analysis of myocardial samples in which immunohistochemistry showed selective loss of protein in the nuclei of cardiac myocytes.
Since the inclusion criterion for the QGELMNA in the 311 cases blind to genotype was consecutive enrollment, the series included both patients and healthy relatives. This may be a limitation for the nonindependence of observations.
Both decreased expression of LMNA gene and variable loss of lamin A/C immunostain in EMB may contribute to the diagnostic algorithm in cardiolaminopathy. In particular, the reduced mRNA expression levels in blood offers to be a potential biomarker in patients for monitoring of the QGELMNA in LMNA mutation carriers.
For an expanded methods and supplementary tables and figure, please see the online version of this paper.
Funded by EU INHERITANCE [Grant Number EU291924]. The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
- Abbreviations and Acronyms
- dilated cardiomyopathy
- endomyocardial biopsy
- nuclear localizing sequence
- premature termination codon
- quantitative gene expression
- receiver-operating characteristic
- Received April 9, 2012.
- Revision received May 23, 2012.
- Accepted May 28, 2012.
- American College of Cardiology Foundation
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