1. 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
• Inherited cardiomyopathies encompass hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy
(ARVC) and are autosomal dominantly inherited with incomplete penetrance.
• 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.
• 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.
Methods
• Gene copy number data from 2,128 individuals referred for cardiomyopathy genetic testing were obtained using a custom-developed, 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.
Introduction
Number (Disease) of Pathogenic Copy Number Variants Reported in HGMD for Cardiomyopathy-Associated Genes
*These genes as well as MYOZ2, NEXN and TTN were reported by Ozge Ceyhan-Birsoy et al. (2015) to harbor clinically significant CNVs.
BAG3
2
(DCM)
2
(DCM)
2
(DCM)
EMD*
4
(Emery-
Dreifuss
muscular
dystrophy)
LMNA* PLN
1 (DCM) 1 (DCM)
CAV3
1
(Rippling Muscle
Disease)
GLA*
23
(Fabry
disease)
MYBPC3* TAZ*DMD
16
(X-linked DCM)
LAMP2*
7
(Danon disease)
PKP2*
9
(ARVC)
1575
(Duchenne/Becker
muscular dystrophy)
• 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 359Kb.
• 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.
ACMG 2016 Poster Presentation
Results
Figure 1. Diagnoses For Probands (n=35) Found To Harbor a Reportable
CNV After Comprehensive Cardiomyopathy Genetic Testing
20.0%
(n=7)
2.9%
(n=1)2.9%
(n=1)2.9%
(n=1)
5.7%
(n=2)
20.0%
(n=7)
17.1%
(n=6)
14.3%
(n=5)
14.3%
(n=5)
Cardiomyopathy, Unspecified
Not Provided
HCM
Asymptomatic
Abnormal Echo and/or EKG
Sudden Cardiac Arrest
Duchenne Muscular Dystrophy
ARVC
DCM

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Results Cont.
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.
• Pathogenic 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).
• 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).
• 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.
• Of 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.
• The individual with a likely pathogenic CNV in PKP2 also harbored a likely pathogenic missense variant in MYH7 detected by NGS.
• Of 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.
Figure 2. Classifications of Reportable CNVs (n= 35) Detected in 2,128 Individuals Referred for Comprehensive Cardiomyopathy Genetic Testing
1
2
2 2
2 2
3
1 1
2
1
1
2
1
1 1
3
2
1
2
11
ABCC9 DMD MYBPC3 PDLIM3 RAF1 TNNI3ACTN2 DSP NEBL PKP2 RYR2 TTNBAG3 LMNA
Pathogenic
DeletionDuplication
Likely Pathogenic Uncertain Significance
NEXN PRKAG2 SOS1