We studied 84 unrelated children (63 boys and 21 girls) with unexplained left ventricular hypertrophy diagnosed at or before 15 years of age (mean age, 6.99±6.12 years) (). Hypertrophy was determined by means of echocardiography () and defined as a mean left ventricular wall thickness of at least 2 SD above the normal value, stratified according to body-mass index or age.1
Echocardiogram Showing Unexplained Asymmetric Left Ventricular Hypertrophy Diagnosed in the First Week of Life during Evaluation of a Boy for Aspiration Pneumonia
The ages of the children at diagnosis ranged from 2 days to 15 years. Initial clinical evaluations were prompted by abnormal physical findings (e.g., murmur or cardiomegaly) identified during routine examinations or by symptoms that were nonspecific (e.g., irritability or lack of feeding in an infant) or suggestive of heart disease (e.g., chest pain). Three children died suddenly from cardiac causes, and in one child this was the presenting manifestation. Over a 5-year follow-up period, 10 children received implantable cardioverter–defibrillators and 5 children underwent cardiac transplantation.
Family histories were positive for cardiomyopathy in 33 children but negative in 51 children who were presumed to have sporadic disease. There were no significant differences with regard to sex distribution, age at presentation, maximum left ventricular wall thickness, contractile function, sudden death, or cardiac transplantation between the group of children with familial disease and the group of children with presumed sporadic cardiomyopathy (). However, significantly more children with a family history of cardiomyopathy received implantable cardioverter–defibrillators (8 of 33 children, vs. 2 of 51 children with no family history; P = 0.007).
Nucleotide sequences encoding eight sarcomere-protein genes (MYH7, MYBPC3, TNNT2, TNNI3, TPM1, MYL3, MYL2
, and ACTC
) and two metabolic genes (PRKAG2
) were determined in each proband. Among children with presumed sporadic cardiomyopathy, 112 sequence variants were identified (Fig. 1 of the Supplementary Appendix
). Seventy-nine synonymous variants or intronic polymorphisms distant from conserved splicing sequences were not predicted to alter the protein structure. Six nonsynonymous MYBPC3
variants (Val158Met, Ser236Gly, Arg326Gln, Val-896Met, Arg1002Trp, and Gln1233Ter) are known polymorphisms.15,17,18
These 85 variants were not studied further.
Among the remaining 27 sequence variants, 3 (MYBPC3
IVS5+6c→t, and TNNT2
IVS14+6c→t) were predicted to alter RNA splice sites or conserved flanking sequences. To assess the effect of these three variants on splicing, we carried out in vitro cell-based assays (Fig. 2 of the Supplementary Appendix
Only one variant, MYBPC3
IVS31+2t→g, disrupted RNA splicing in a way that would be predicted to alter protein structure (Fig. 2 of the Supplementary Appendix
), and it is thus similar to an established MYBPC3
splice-site mutation that causes familial hypertrophic cardiomyopathy.21
We therefore evaluated MYBPC3
IVS31+2t→g, but not MYL3
IVS5+6c→t or TNNT2
IVS14+6c→t, along with the other 24 variants predicted to affect protein structure and function.
We observed 25 unique, nonsynonymous sequence variants in MYH7, MYBPC3, TNNT2, TNNI3, ACTC
, and PRKAG2
(, and Fig. 1 of the Supplementary Appendix
) in children with presumed sporadic cardiomyopathy. We did not observe associations between variant residues in the three other genes associated with hypertrophic cardiomyopathy — TPM1
, and MYL3
— and childhood-onset left ventricular hypertrophy. Each nonsynonymous sequence variant was independently confirmed by restriction-enzyme digestion of PCR-amplified sequences. Fourteen of the 25 variants () have been reported to cause familial hypertrophic cardiomyopathy15,18,22–33
; 12 of these variants also encode missense residues, 1 is predicted to delete an amino acid, and 1 is predicted to prematurely terminate protein translation.
Gene Mutations in Patients with Childhood-Onset Cardiac Hypertrophy and Presumed Sporadic Cardiomyopathy.*
Eleven sequence variants were not previously reported: MYH7
Arg97Cys, and PRKAG2
His530Arg. Nine of these previously unreported variants caused missense changes and altered amino acid residues that are highly conserved during mammalian evolution (Fig. 3 of the Supplementary Appendix
), one variant deleted a highly conserved glutamic acid residue in TNNT2
, and one variant altered MYBPC3
splicing (detailed above). Each of these 11 previously unreported variants was absent in 180 unrelated persons matched by ancestral origin to the subjects (data not shown) and in more than 1000 chromosomes of unaffected persons (unpublished data). Furthermore, sequence analyses of these eight sarcomere-protein genes in 34 persons without left ventricular hypertrophy revealed no variants.34
On the basis of a chi-square analysis, the probability that 25 nonsynonymous sequence variants occurred by chance in 51 cases of pediatric-onset left ventricular hypertrophy is less than 1 in 500,000. We conclude that each of these previously unreported variants, like the 14 mutations known to cause hypertrophic cardiomyopathy, caused left ventricular hypertrophy in these children.
Three of the 25 children with presumed sporadic cardiomyopathy who had mutations (12%) had two different sarcomere-gene mutations: MYH7
Arg663His and Val763Met; MYH7
Arg787Cys and ACTC
Arg97Cys; and MYBPC3
Thr1028Ser and MYBPC3
IVS31+2t→g. We observed the MYBPC3
IVS31+2t→g mutation in isolation in another child; others27
have reported MYH7
Arg663His in isolation as the cause of hypertrophic cardiomyopathy. To determine whether sporadic left ventricular hypertrophy in the children reflected new mutational events, we genotyped the parents of 11 probands () and confirmed parental relationships using 22 polymorphic microsatellite markers (data not shown). Four mutations (MYH7
Arg92Gln, and TNNT2
Glu96del) were absent from both parents, confirming that new mutations accounted for sporadic cardiomyopathy in these children. In contrast, mutations found in seven children were also present in the parents, indicating that these mutations were inherited (). Clinical studies revealed cardiomyopathy in one parent, were not performed in two parents, and showed no abnormalities in five parents, two of whom carried one of the two MYBPC3
mutations carried by their compound heterozygous (Thr1028Ser and IVS31+2t→g) affected child.
Family Pedigrees of 11 Patients with Childhood-Onset Idiopathic Cardiac Hypertrophy and No Family History of Cardiomyopathy
To determine the parental origins of chromosomes for new mutations, closely linked polymorphisms were analyzed. Haplotype analyses were not informative in two subjects (with mutations MYH7
Lys146Asn and TNNT2
Glu96del). Haplotype analyses indicated that the new mutation MYH7
Glu924Lys arose on a maternal chromosome (Fig. 4A of the Supplementary Appendix
), and the new mutation TNNT2
Arg92Gln arose on a paternal chromosome (Fig. 4B of the Supplementary Appendix
We also sequenced genes in 33 patients who had childhood-onset cardiac hypertrophy and family histories of cardiomyopathy but who had not undergone previous genetic studies. Twenty-two nonsynonymous sequence variants (Table 3 of the Supplementary Appendix
) were identified in 21 patients (64%); each variant altered a sarcomere-protein gene: MYH7
(in 9 patients), MYBPC3
(in 7 patients), TNNT2
(in 2 patients), TNNI3
(in 1 patient), TPM1
(in 2 patients), and MYL3
(in 1 patient). No variants were found in ACTC
. Fifteen sequence variants were previously reported as cardiomyopathy mutations (Table 3 of the Supplementary Appendix
). The remaining seven variants (MYH7
Ser215Leu, and MYL3
Met173Val) were absent from 180 unrelated, ancestrally matched persons (data not shown) and from more than 1000 chromosomes of unaffected persons (unpublished data). Each of these new variants altered amino acid residues that are highly conserved throughout mammalian evolution (Fig. 5 of the Supplementary Appendix
), predicting that the missense residue alters protein structure.
Among mutation-positive children with familial disease, three (14%) had compound mutations (MYBPC3 Arg502Trp and MYBPC3 Ser858Asn; MYBPC3 Arg495Gln and TNNI3 Arg141Gln; and MYBPC3 Ile154Thr and MYBPC3 Asp605del).