COPY NUMBER VARIANTS IN CARDIOMYOPATHY-ASSOCIATED
GENES
Daniela Macaya, PhD, FACMG; Rebecca Latimer, MMSc, CGC; Gabriele Richard, MD, FACMG; Shelley Patrick, MS, CGC; Christian Antolik, PhD, FACMG
GeneDx, Gaithersburg, Maryland, USA
Introduction
•Inherited cardiomyopathies encompass hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM),
and arrhythmogenic right ventricular cardiomyopathy (ARVC) and are autosomal dominantly inherited with
incomplete penetrance.
Number (Disease) of Pathogenic Copy Number Variants Reported in HGMD for Cardiomyopathy-Associated Genes
1575
(Duchenne/Becker
muscular dystrophy)
•Copy number variant (CNV) analysis of cardiomyopathy-associated genes is often included in clinical testing.
Despite loss-of-function being a known disease mechanism for some cardiomyopathy genes, the clinical utility
of this analysis for cardiomyopathy as a whole has not been well-defined.
23
(Fabry disease)
16(X-linked
DCM)
4 (Emery-Dreifuss
2
1 (Rippling
muscular dystrophy)
(DCM) Muscle
Disease)
•Our diagnostic laboratory performs molecular testing for cardiomyopathies using a multigene panel with
combined next generation sequencing (NGS) and CNV analysis. This panel includes copy number analysis of
60 nuclear genes. CNVs associated with cardiomyopathy have been reported in the Human Genome Mutation
Database (HGMD) in 11 of these genes. In addition to the CNVs reported in HGMD, a recent study (Ozge
Ceyhan-Birsoy et al., 2015) reported clinically significant CNVs in EMD, GLA, LAMP2, LMNA, MYBPC3, MYOZ2,
NEXN, PKP2, TAZ and TTN.
BAG3
CAV3
DMD
EMD*
GLA*
7
(Danon
disease)
LAMP2*
2
(DCM)
2
(DCM)
LMNA*
MYBPC3*
9
(ARVC)
PKP2*
1
(DCM)
1 (DCM)
PLN
TAZ*
*These genes as well as MYOZ2, NEXN and TTN were reported by Ozge Ceyhan-Birsoy et al. (2015) to harbor clinically significant CNVs.
Methods
•Gene copy number data from 2,128 individuals referred for
cardiomyopathy genetic testing were obtained using a customdeveloped, targeted oligonucleotide array and gene-specific filtering with
Cartagenia Lab Bench for 60 nuclear cardiomyopathy genes.
•Reportable CNVs were classified as either pathogenic, likely pathogenic,
or of uncertain significance based on review of internal and external data
concerning presence/absence in disease and control populations, family
data, in silico predictions, and gene-specific knowledge about function
and role in disease.
•Data was abstracted for probands found to harbor a reportable
CNV and included variant classification, location and size, as well as
demographic and clinical information.
Results
•Of 2,128 individuals referred for testing, 35 (1.6%) unrelated individuals harbored a reportable CNV, 33 were
unique. Nineteen (54.3%) were deletions and 16 (45.7%) were duplications ranging in size from 0.5 Kb to 359 Kb.
•The majority (n= 24, 68.6%) of the 35 were referred for genetic testing due to a personal history of
cardiomyopathy (Figure 1). Twenty-one probands had at least one relative affected with the either the same or
suspicious phenotype (i.e. sudden cardiac arrest/sudden unexplained death, congestive heart failure and/or
abnormal EKG/Echo), four individuals had no family history of cardiac disease, and 10 referrals did not provide
family history information. Approximately thirty-one percent (11/35) of the referrals did not provide ancestry
information. Forty percent (14/35) of the probands were Caucasian, and the remaining (n= 10, 28.6%) reported
either African American or Hispanic ancestry. The number of male (n= 16, 45.7%) and female (n= 19, 54.3%)
probands was similar.
Figure 1. Diagnoses For Probands (n=35) Found To Harbor a Reportable CNV After Comprehensive Cardiomyopathy
Genetic Testing
2.9%
2.9%
2.9% (n=1) (n=1)
(n=1)
5.7%
(n=1)
Cardiomyopathy,
Unspecified
Not
Provided
20.0%
(n=7)
Asymptomatic
HCM
14.3%
Sudden Cardiac Arrest
Abnormal Echo and/or EKG
(n=5)
• P
athogenic CNVs: Ten CNVs (28.6%) were classified as pathogenic, all of which were partial deletions. These
pathogenic gene deletions represented 1.9% of all pathogenic findings (n=518) obtained by cardiomyopathy
panel testing. Six of these 10 CNVs were terminal deletions extending into the 5’ or 3’ UTR, and the remainder
(n=4) were intragenic deletions of one to ten exons. Of note, five of six CNVs involving the ARVC-associated
genes DSP and PKP2 were pathogenic or likely pathogenic (Figure 2).
• O
f the 10 pathogenic CNVs, five co-occurred with at least one sequence variant of uncertain significance.
Additionally, one individual with a pathogenic CNV in MYBPC3 was also heterozygous for a pathogenic
nonsense variant in DSP detected by NGS.
• T
he individual with a likely pathogenic CNV in PKP2 also harbored a likely pathogenic missense variant in
MYH7 detected by NGS.
• O
f the 24 CNVs of uncertain significance, 16 co-occurred with at least one sequence variant, of which only
one was pathogenic and explained the patient’s phenotype.
Duchenne Muscular
Dystrophy
ARVC
DCM
17.1%
(n=6)
Figure 2. Classifications of Reportable CNVs (n= 35) Detected in 2,128 Individuals Referred for Comprehensive Cardiomyopathy
Genetic Testing
Duplication
•
Co-occurring reportable variants: A high proportion (n= 23, 65.7%) of individuals with a reportable CNV also
harbored at least one reportable variant detected by NGS.
14.3%
(n=5)
Deletion
•
CNVs of uncertain significance: Twenty-four CNVs (68.6%) of uncertain clinical significance were identified, 15
were duplications with the majority (n=9) under 100Kb (Figure 2).
20.0%
(n=7)
1
2
1
ABCC9
2
2
ACTN2
BAG3
1
2
2
DMD
DSP
1
1
MYBPC3
NEBL
3
LMNA
Pathogenic
2
NEXN
Likely Pathogenic
1
PDLIM3
1
3
1
1
2
1
2
2
PKP2
1
PRKAG2
RAF1
RYR2
SOS1
TNNI3
TTN
Uncertain Significance
Conclusions
•These data demonstrate that pathogenic CNVs in genes associated with inherited cardiomyopathy account
for almost 2% of reportable diagnostic results, underscoring the clinical utility of gene copy number analysis as
part of molecular diagnostic testing panels. Overall, clearly pathogenic CNVs in our large data set were found in
0.5% (10/2128) of patients tested, consistent with a previously published pathogenic CNV rate of 0.8% (4/505) in
a cohort with HCM and clinically significant CNV rate of 0.6% (9/1425) in a cohort with cardiomyopathies (Lopes
et al., 2015; Ozge Ceyhan-Borsoy et al., 2015).
•Almost half of the reported CNVs in this study were duplications of uncertain significance, many of which likely
occurred in tandem and may disrupt the open reading frame, but further evidence is needed to determine their
pathogenicity.
•The known loss-of-function mechanism for DMD, DSP, LMNA, MYBPC3, and PKP2 supports our findings
of pathogenic CNVs in these genes. Albeit rare, the identification of pathogenic CNVs has important clinical
implications for the affected patients and their families.
References
1.Ozge Ceyhan-Borsoy et al. Next generation sequencing-based copy number analysis reveals low prevalence of deletions and duplications in 46 genes associated with
genetic cardiomyopathies. Mol Genet Genomic Med. Epub 2015 Dec 16; http://dx.doi.org/10.1002/mgg3.187.
2.Lopes et al. Use of high-throughput targeted exome-sequencing to screen for copy number variation in hypertrophic cardiomyopathy. Eur J Med Genet. 2015
Nov;58(11):611-6.
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