Assessment of LMNA Copy Number Variation in 58 Probands with Dilated Cardiomyopathy

Abstract 

The contribution of copy number variation (CNV) to dilated cardiomyopathy (DCM) is unknown. However, estimates have suggested that CNVs could constitute 15% of mutations underlying Mendelian disease. This is of particular relevance to DCM, where only approximately 35% of genetic cause has been identified. We have previously reported 19 point mutations in LMNA, the gene encoding Lamin A/C, in a cohort of 324 unrelated DCM probands (5.9%), making it the most common genetic cause of DCM. Recently a large deletion was reported in LMNA in 1 of 25 DCM probands. To further assess the contribution of CNVs in LMNA cardiomyopathy, we used Multiplex Ligation Probe Amplification (MLPA) to screen for large deletions and duplications in 58 DCM probands negative for point mutations in LMNA. Despite excellent quality control and robust MLPA results, our study failed to identify any deletions or duplications. We conclude that at least for LMNA, point mutations are the major source of DCM causation. Clin Trans Sci 2011; Volume 4: 351–352

Introduction

Dilated cardiomyopathy (DCM) is a primary myocardial disease characterized by left ventricular enlargement and systolic dysfunction. The DCM term used herein, also known as idiopathic DCM, implies nonsyndromic DCM with other known causes ruled out. To date, rare variants in 32 autosomal and two X‐linked genes have been associated with DCM. The frequency of rare variants in any one DCM gene is low (<1% to 6%), and a genetic cause has been identified in only approximately 35% of familial cases. ¹ Notably, these frequencies have been estimated from sequencing the coding regions of these genes, a method that would fail to identify large (>1 kb) deletions, duplications, or copy number variation (CNV).

Recent findings suggest that CNV does indeed contribute to DCM and other Mendelian cardiovascular disease ² , ³ , ⁴ but its contribution to DCM compared to point mutations is unknown. Determination of CNVs requires costly and time‐consuming laboratory assays. Cost‐effective use of resources for predictive screening will require systematic determination of the frequency of this type of genetic variation in cardiovascular disease.

In our resequencing studies of the most common known DCM genes, ^{3 , 5} , ⁶ , ⁷ , ⁸ , ⁹ LMNA, the gene encoding Lamin A/C, had the highest reported fraction of probands carrying single base mutations (19 of 324 DCM subjects, 5.9%). ⁵ A study of 25 DCM patients with conduction disorder ² reported a proband with a heterozygous deletion spanning LMNA exons 3–12, suggesting LMNA CNVs may account for a similar amount of genetic cause as point mutations.

In this study, we aimed to assess the frequency of large deletions and duplications in LMNA in 58 probands, a subset of DCM patients who were point mutation negative in LMNA and for most in 15 other known DCM genes. ^{3 , 5} , ⁶ , ⁷ , ⁸ , ⁹

Methods

Patient population

Written, informed consent was obtained from all subjects, and the institutional review boards at the Oregon Health and Science University and the University of Miami approved the study. Case selection for this study was based upon analysis of individual and discrete data elements, such as conduction system disease, presence of arrhythmia, pacemaker or ICD, at any point in the subject’s disease course, rather than a clinical scenario of progressive conduction system disease, followed by arrhythmia and DCM, consistent with LMNA cardiomyopathy. We screened a total of 58 subjects with DCM, of which 96.6% were white (53/56 non‐Hispanic, 3/56 Hispanic) and 3.4% were African American. Clinical details are shown ( Table 1 ).

Table 1.

Clinical characteristics of study sample.

Demographics
Average age at DCM diagnosis	43.6 ±13.4 years, range 16–76
Familial dilated cardiomyopathy	N = 32 (55%)
Previous Resequencing Studies3,5–9
LMNA point mutation negative	N = 58 (100%)
Point mutation negative in 15 other DCM genes	N = 44 (76%)
Clinical Features
Arrythmia/Conduction System Disease
Conduction system disorder	N = 58 (100%)
Any arrhythmia	N = 45 (78%)
Pacemaker	N = 13 (22%)
Implantable cardiac defibrillator	N = 38 (66%)
Sudden death during disease course	N = 14 (24%)
Myocardial Failure
Heart failure	N = 52 (90%)
Heart transplant or ventricular assist device	N = 13 (22%)
Death (without transplant or ventricular assist device)	N = 10 (17%)

MLPA assay

We used mulitplex‐ligation probe amplification (kit P048SALSA MLPA KIT P048‐B1, lot 1108, MRC Holland, Amsterdam, The Netherlands) to screen for deletions and duplications in each exon of the LMNA gene. The probe mix contained two probes in exon 1 and one probe in each of exons 2–12. Reactions were performed as per the manufacturer’s instructions, (MRC‐Holland). For each sample, 2 μL MLPA amplification product was run on a 3130XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) in 10 μL HIDI formamide and 0.5 μL GeneScan 500 ROX internal size standard (Applied Biosystems). Peak area data was analyzed in Coffalyzer MLPA‐DAT (MRC‐Holland).

Results

In addition to gene specific and control probes, the MLPA assay contained a number of quality control (QC) probes for each stage of the reaction. Ligation dependent and denaturation QC probes were present for all 58 samples. Median absolute deviation of control probes ranged 0.02–0.1 indicating the assay was reliable.

Normalized ratios of test probes to control probes in DCM subjects relative to three reference individuals were expected to be approximately 1 for normal copy number, approximately 0.5 for heterozygous deletions, and approximately 1.5 for heterozygous duplications. Ratios for all 58 DCM subjects were approximately 1, indicative of normal copy number.

Discussion

The Human Genome Mutation database reports over 100,000 known mutations underlying human inherited diseases from 3,960 different genes, the majority of which are single base coding mutations. This figure is likely to represent only a small proportion of the clinically relevant genetic variants. Estimates suggest that copy number variants may constitute 15% of all mutations underlying Mendelian disease. ¹⁰ This could be particularly relevant to DCM, where approximately 65% of the genetic cause is unaccounted for and studies of CNV are so far unaccomplished.

The first report of CNV across LMNA screened 25 DCM patients, identifying a single individual with a large heterozygous deletion. ² With a frequency of 1/25 (approximately 4%), these data suggested that at least for LMNA, CNV could account for a similar proportion of genetic cause as point mutation. However, in a larger cohort of 58 DCM patients, using the same molecular assay as the original study, we failed to identify any large deletions or duplications in the LMNA gene. Combining the data from these two studies suggests that CNV in LMNA has a frequency of no more than 1/83 (approximately 1%), which is significantly less than that accounted for by point mutations (approximately 6%).

Of note, patients differed between these studies. The Gupta study ² used a patient group more likely to be enriched for LMNA mutations based on the presence of either major cardiomyocyte nuclear abnormalities (eight patients), or nonspecific nuclear abnormalities (17 patients). In addition to a single deletion, they also identified three patients carrying LMNA point mutations, a fraction of 4/25 (16% CNV or point mutation, 12% for point mutation only). This is significantly enriched compared to the 5.9% fraction of DCM probands positive for LMNA mutations in our prior study, which were detected from 324 probands with DCM unselected for nuclear abnormalities or for conduction system disease commonly seen with LMNA cardiomyopathy. ⁵

The translational relevance of this finding is that subjects with DCM are more likely to carry point mutations than deletions or duplications in the LMNA gene. However, we hesitate to generalize this finding to the many other genes known to cause DCM, as CNV studies have not been performed in most genes associated with DCM. At this time, point mutations and CNV are assessed with different molecular assays and platforms. Future development of next generation sequencing technologies to simultaneously determine both point mutation and CNV from the same genetic platform will provide major advancement to the field.

Conflict of Interest

The authors report no conflict of interest.