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Online Appendix for the following JACC: Cardiovascular Imaging article
TITLE: CMR-Verified Interstitial Myocardial Fibrosis as a Marker of Subclinical Cardiac
Involvement in LMNA Mutation Carriers
AUTHORS: Marianna Fontana, MD, Andrea Barison, MD, Nicoletta Botto, PHD, Luca Panchetti,
MD, Giulia Ricci, MD, Matteo Milanesi, PHD, Roberta Poletti, MD, Vincenzo Positano, MSc,
Gabriele Siciliano, MD, PHD, Claudio Passino, MD, PHD, Massimo Lombardi, MD, Michele
Emdin, MD, PHD, Pier Giorgio Masci, MD
____________________________________________________________
APPENDIX
Genetic analysis
Mutation screening was carried out with genomic DNA samples from the probands by screening of
the 12 coding exons of LMNA/C gene (GenBank accession n. NM_170707.2) amplified by
polymerase-chain reaction. Primers encompassing the protein coding region of exons and the
immediate intronic regions essential for splicing were used, as described previously. Purified
polymerase-chain reaction products were sequenced in both directions using a capillary sequencer
CEQ 8800 (Beckman Coulter-Germany). All detected variants, except for duplication, were
confirmed and allele frequencies assessed in controls by polymerase-chain reaction-based
restriction fragment length polymorphism analysis using specific endonucleases (TauI for
p.R189W, AvaII for p.D461Y, DdeI for p.K117R), with the exception of p.R110S assessed by
direct sequencing. After giving informed consent, relatives of LMNA/C-mutation carriers received
genetic counseling and clinical screening, and underwent peripheral blood sampling for genetic
testing consisting of bidirectional analysis for the exon containing the proband’s mutations.
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We found 5 missense mutations: R110S, K117R, R189W, D461Y, R190W; 1 frame shifting
mutation: A368_Y376dup fsX112; 1 splicing mutation: c.1608+1G>T. Six mutations were novel,
none of these variants was identified in 100 chromosomes of ethnically-matched, healthy
individuals (≥30 years) who were randomly selected from our control genomic store nor previously
published in literature as benign polymorphism, indicating they were not common variants. All
novel missense mutations affect amino acids with high degree of conservation throughout
evolution. One mutation (R190W) was known to cause DCM (1). Remarkably, four of them
clustered in the coil 1b of alpha-helical rod domain affecting LMNA/C, whereas one mutation
(D461Y) was located in an immunoglobulin-like domain at C-terminal globular tail region. The
c.1608+1G>T mutation affects the first nucleotide of the 5' splice donor site (that includes an
almost invariant sequence GU at the 5' end of the intron) with a high degree of conservation
throughout evolution. The potential effect of the discovered mutation was assessed using three
different methods for prediction of splice sites, NetGene2, ASSP and HSF-SSF, and the results
obtained suggest that the c.1608+1G>T mutation could be involved in generating aberrant
transcripts.
Cardiovascular Magnetic Resonance
Study participants were examined by a 1.5-T unit (CVi, GE-Healthcare, Milwaukee-USA) using
dedicated cardiac software, phased-array surface receiver coil and vectocardiogram triggering.
Ventricular function was assessed by breath-hold steady-state free-precession cine imaging in
cardiac short-axis, vertical and horizontal long-axis. In cardiac short-axis, ventricles were
completely encompassed by contiguous 8 mm thick slices. Sequence parameters were: field-ofview: 380-400mm, repetition/echo time: 3.2/1.6ms, flip angle: 60°, matrix: 224x192, phases: 30.
For the determination of T1 value of myocardium and blood cavity, a single mid-ventricular shortaxis modified-cine inversion-recovery (MCine-IR) sequence was performed before and at fixed
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time intervals (5-min, 10-min, 15-min) after the administration of a bolus of contrast agent
(Gadodiamide-OMNISCAN, 0.2 mmol/kg). The sequence is described in the details further.
Briefly, MCine-IR consisted of non-selective adiabatic inversion pulse applied at the R-wave of
ECG and followed by cine segmented gradient-echo acquisition extended to the subsequent 4-6RR
intervals for pre-contrast imaging or 2-RR intervals for post-contrast imaging. Sequence
parameters: field-of-view: 380-400mm, repetition/echo time: 6/2.8 ms, flip angle: 8°, matrix:
224x192, phases: 40. LGE imaging was performed between 10 and 20 minutes after contrast agent
administration using segmented T1-weighted gradient-echo inversion-recovery pulse sequence. In
short-axis orientation, LV was completely encompassed by contiguous 8-mm thick slices. Images
were also acquired in vertical and horizontal long-axis views. Inversion time was individually
adapted to suppress the signal of normal remote myocardium (220 to 300 ms). Sequences
parameters were: field-of-view: 380-400mm, slice thickness: 8mm, repetition/echo time: 4.6/1.3ms,
flip angle: 20°, matrix: 256x192.
All CMR studies were analyzed off-line using a workstation (Advantage Workstation, GE
Healthcare, Milwaukee-USA) with dedicated software (MASS 6.1, Medis, Leiden-Netherlands) by
one experienced operator unaware of clinical data. Using the stack of short-axis cine images, LV
volumes, mass and global function were determined. LV volumes and mass were normalized to
body-surface-area. LV dysfunction and dilatation were considered present if LV ejection-fraction
and end-diastolic volume resulted beyond the 99th percentile of normal values corrected for age and
body-surface-area (2). Presence of LGE was determined on post-contrast images, and its extent was
automatically calculated on short-axis images by adopting a signal intensity threshold >6SD above
the mean signal intensity of the remote normal myocardium (3).
For T1-mapping analysis a custom-written software (HIPPO-SW®) developed in IDL 7.1 was
utilized. Briefly, LV endocardial and epicardial borders were traced in the single midventricular
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short-axis slice, which was divided into 6 equiangular segments starting from inferior right
ventricular-LV insertion. Signal intensity vs time curve for each segment and blood pool was
utilized to determine segmental T1 and blood pool values (and their reciprocal, R1) by adopting an
exponential fitting, (Figure 1). After confirmation that a steady state equilibrium of gadolinium
between the plasma and myocardium was reached 10 minutes after contrast agent administration,
R1 measurements performed at 10 and 15 minutes were then used to calculate the partition
coefficient of gadolinium (λGd) from the slope of the linear regression line for the measured values
of R1 (myocardium) vs R1 (blood) by exploiting the linear relationship between change in
relaxivity and gadolinium concentration. After correction for myocardial/blood relaxivity ratio (0.8
at 1.5 Tesla), corrected partition coefficient of gadolinium (cλGd) was calculated as λGd /0.8. To
determine myocardial ECV, we used the following formula ECV=cλGd x (1-hematocrit) – Vp,
where Vp is the myocardial plasma volume fraction (assumed to be a constant 0.045, reflecting
capillary density), (4,5).
Modified Cine Inversion Recovery (MCine-IR) sequence
The MCine-IR principle is illustrated in the pulse sequence of Figure 2. This sequence consists of a
non-selective adiabatic inversion pulse applied immediately after the ECG R-wave trigger. The
inversion pulse is followed by a Cine (segmented k-space) acquisition where each cardiac phase
experiences a different time delay after the inversion pulse and thus a different T1 weighting.
However, while in the original cine acquisition was limited to the first heart cycle following the
inversion and the second cycle was left for T1 relaxation (6-8) and Cine acquisition was made from
the subsequent R wave, in the our MCine-IR the acquisition is extended to further heart cycles
allowing for extended recovery of longitudinal magnetization. This allows pre-contrast myocardium
and blood to fully recover the longitudinal magnetization during Cine acquisition. Linear
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retrospective interpolation was performed on the Cine data acquired in a single breath-hold, to fully
cover multiple cardiac R-R intervals. The number of heart beats used for the acquisition is given by:
 

TR
eff
ifmod
TR
,Δ

0

eff
RR
RR
Δ
N


RR
TR
eff

1 ifmod
TR
,Δ

0
eff
RR
Δ 
 RR
 




(1)
where | | denotes integer division and mod( ) the remainder of the integer division. ΔRR is the RR
interval of the subject in ms and TReff is the effective TR between two successive inversion pulses.
The addition of +1 in the formula avoids any underestimation of TReff by rounding up its value to
the next heart beat. The true effective TR of MCine-IR is given by Nrr · ΔRR.
In our study lamin A/C (LMNA) mutation carriers and normal controls were scanned by MCine-IR
using a non-selective adiabatic inversion pulse applied immediately after the ECG R-wave and the
inversion pulse is followed by a cine acquisition (40 frames) extended to the subsequent 4-6 RR
intervals (depending of the subject’s heart rate) for pre-contrast imaging or subsequent 2 RR for
post-contrast imaging allowing: 1) full pre- and post-contrast longitudinal magnetization recovery
of myocardium and blood before the application of the next inversion pulse; 2) complete sampling
throughout of pre- and post-contrast longitudinal magnetization recovery of myocardium and blood
cavity. In order to accomplish a correct estimation of myocardial ECV, a steady state equilibrium of
gadolinium between myocardium and blood cavity is required. In this condition, the gadolinium
partition coefficient (λGd) remains constant over time (5). We measured myocardial and blood
cavity T1 values at 5, 10 and 15 minutes after gadolinium injection (Gadodiamide-OMNISCAN,
0.2 mmol/kg) and then we derived λGd for each time point. In the overall study population, the
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steady state was reached at 10 minutes after bolus contrast injection (Figure 3). This result is nicely
concordant with recent findings indicating that the steady state between myocardium and blood
cavity is reached 12 minutes after a bolus of 0.2 mmol/kg of gadolinium-chelate and maintained up
to 50 minutes, similarly to what occurs with continuous bolus administration (4, 9).
Supplemental references
1. Arbustini E, Pilotto A, Repetto A, et al. Autosomal dominant dilated cardiomyopathy with
atrioventricular block: a lamin A/C defect-related disease. J Am Coll Cardiol. 2002;39:981-90
2. Maceira AM, Prasad SK, Khan M, Pennell DJ. Normalized left ventricular systolic and diastolic
function by steady state free precession cardiovascular magnetic resonance. J Cardiovasc Magn
Reson. 2006;8(3):417-26.
3. Bondarenko O, Beek AM, Hofman MB, et al. Standardizing the definition of hyperenhancement
in the quantitative assessment of infarct size and myocardial viability using delayed contrastenhanced CMR. J Cardiovasc Magn Reson. 2005;7:481-5.
4. Schelbert EB, Testa SM, Meier CG et al. Myocardial extravascular extracellular volume fraction
measurement by gadolinium CMR in humans: slow infusion versus bolus. J Cardiovasc Magn
Reson. 2011;4;13-16.
5. Jerosch-Herold M, Sheridan DC, Kushner JD, et al. Cardiac magnetic resonance imaging of
myocardial contrast uptake and blood flow in patients affected with idiopathic or familial dilated
cardiomyopathy. Am J Physiol Heart Circ Physiol. 2008;295:H1234-H1242.
6. Gupta A, Lee V S, Chung Y-C, Babb J S, Simonetti O P. Myocardial infarction: optimization of
inversion times at delayed contrast-enhanced MR imaging. Radiology 2004; 233:921-926
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7. Ho V B, Hood M N, Montequin M, Foo T K. Cine Inversion Recovery (IR): Rapid tool for
optimized myocardial delayed enhancement imaging. In: Proceedings of the 13th Annual Meeting of
the ISMRM 2005: p. 1675.
8. Goldfarb J W, Mathew S T, Reichek N. Quantitative breath-hold monitoring of myocardial
gadolinium enhancement using inversion recovery TrueFISP. Magn Reson Med 2005; 53:367-371
9. Klein C, Nekolla S G, Balbach T, Schnackenburg B, Nagel E, Eckart F, Schwaiger M. The
Influence of myocardial blood flow and volume of distribution on late Gd-DTPA kinetics in
ischemic heart failure. J Magn Reson Imaging 2004; 20:588-594.
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Supplemental Figures
Figure 1. Differences between LMNA mutation carriers and controls. Box plots show median,
quartile, and extreme values of left ventricular (LV) end-diastolic volume (A), ejection fraction (B),
and myocardial extracellular/extravascular volume (ECV) (C) in LMNA mutation carriers and
healthy controls.
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Figure 2. A selected series of MCine-IR mid-ventricular short-axis images in a LMNA-mutation
carrier with LV dysfunction acquired 15 minutes after contrast-administration (upper panel). The
graph (bottom panel) shows the signal intensity of blood cavity and myocardium against the time
delay after the nonslice-selective inversion pulse. The continuous lines represent least-squares fits
to the data points.
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Figure 3. MCine-IR pulse sequence. A Cine acquisition scheme is performed between two
consecutive non selective adiabatic 180° inversion pulses. The number of heart beats between the
two inversion pulses is determined according to Eq. [1] by the chosen TReff and patients heart rate.
The number of inversion pulses, i.e. the number of MCine-IR module repetitions, is controlled
through the phase encoding steps and the views per segments as for a standard cine sequence.
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Figure 4. Gadolinium partition coefficient comparison between 5, 10 and 15 min after bolus
injection in the overall population (n=53, comprising 16 controls, 19 lamin A/C mutation carriers
and 18 dilated cardiomyopathy patients). †P<0.05; *P=NS. The Friedman’s test was used to test
differences between the three time points (multiple comparison), whereas the difference between
pair of measures was assessed by Wilcoxon’s test.
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