Hypertrophic cardiomyopathy (HCM; MIM 192600) is defined clinically by the presence of a hypertrophic, non-dilated left ventricle in the absence of increased external haemodynamic load and, histologically, by cardiomyocyte hypertrophy, myofibre disarray and interstitial fibrosis. Global left ventricular systolic function, as indicated by ejection fraction, is often preserved or supranormal while diastolic functional impairment results in delayed relaxation, increased filling pressures and heart failure symptoms.1 The clinical outcomes of HCM are highly variable and affected subjects in the same family often exhibit a wide range of clinical manifestations from early-onset, severe hypertrophy to milder disease presenting during adulthood.2

HCM is a common disorder affecting 1 in 500 of the population and, while generally inherited in an autosomal dominant fashion, it also occurs sporadically.3 4 Mutations within multiple genes encoding proteins which function within the sarcomere have been identified.5 Several studies have recognised the gene-encoding myosin-binding protein C (MYBPC3) as a common cause being the most frequently mutated gene in many populations.6^–11 Heterozygous mutations in this gene seem to be associated with a later-onset hypertrophy and a favourable clinical course.12

Despite the prevalence of MYBPC3 mutations, only a few cases of subjects with homozygous or compound heterozygous mutation have been reported. In this report we have identified the presence of a splice site mutation, predicted to lead to premature termination of the MYBPC3 polypeptide product. This mutation is at a frequency amongst the Old Order Amish that results in severely affected offspring of asymptomatic parents with a much higher prevalence than found in the general population. This unique culturally isolated population has been recognised to be at increased risk for a number of autosomal recessive disorders owing to the inheritance of two copies of ancestral founder mutations.13^–15 We report the identification of multiple cases of infantile disease associated with homozygous MYBPC3 mutation and their clinical and pathological findings.

METHODS

Case ascertainment

All the affected subjects were identified during the course of routine paediatric care and referred for cardiology evaluation owing to clinical symptoms of cardiac dysfunction. Parents of affected infants gave their informed consent to participate in the study, which was approved by the local research ethics committee.

Genome scan

A total of 10 affected infants (four male, six female) were identified and DNA was extracted, using the Qiagen QIAamp DNA extraction kit (Qiagen, Crawley, UK), from either cord blood, venous blood or placental tissue samples for genotyping. Medical information about the affected subjects was obtained from their families or their primary care doctors or cardiologists. A genome-wide screen was performed using Affymetrix SNP 10K arrays (Medical Research Council Geneservice, Cambridge, UK). DNA from four affected children and that of their parents were used in the initial analysis. Microsatellite marker confirmation of homozygosity across this region was performed by PCR and polyacrylamide gel electrophoresis, as previously described.15

Mutation analysis

Primers for mutation analysis (sequences available on request) were positioned flanking each coding exon and associated splice junctions of the MYBPC3 gene for amplification by PCR using 50 ng genomic DNA using 10 pmol of each primer (as described in Simpson et al, 200414). Subsequent bidirectional sequencing was performed using the ABI terminator cycle sequencing kit (version 3.1; Applied Biosystems, Cheshire, UK). The variant in intron 30 of the gene, which creates an Nla IV restriction site, was verified by restriction digest analysis; PCR products were subsequently digested using 1 unit of restriction enzyme Nla IV and visualised on agarose gels. This variant was absent in 100 control chromosomes, suggesting that it is not a polymorphism.

RESULTS

Clinical evaluation

Diagnostic cardiology testing for most affected subjects included electrocardiograms and echocardiograms. Standard clinical management included the use of mechanical ventilatory support, intravenous fluids and a range of positive inotropic and vasodilator drugs. Affected children all had clinical deterioration despite maximal medical treatment. Table 1 summarises the clinical presentation, echocardiographic findings and clinical course of each case. Of note, five patients were ultimately treated successfully with orthotopic heart transplantation. An additional five siblings died during infancy and were not included in the current genetic analysis. The clinical status of the parents of the affected children has not been systematically assessed. However, they are typically but not uniformly asymptomatic in their twenties. One of the parents who did present with cardiac abnormalities had a mild form of asymmetric septal hypertrophy with a mild degree of left ventricular outflow tract obstruction.

Representative histology

Postmortem evaluation of a representative case showed a globular and slightly enlarged heart with normal cardiac dimensions for age. On surface examination no evidence of ischaemic change or fibrosis was evident. The coronary arteries did not show any structural alterations. Histological assessment using H&E stain showed widespread evidence of myofibre disarray in both right and left ventricular myocardium (figs 1A and B). Myocytes intersected each other randomly with adjacent longitudinally and transversely oriented myocytes, areas of whorl formation and evidence of Y-shaped branching myocytes.

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Figure 1 Microscopic evaluation of heart samples showed myocardial disarray characterised by disordered myocytes that branch at various angles with foci of whorled appearance (A, B). There was marked difference in size of intersecting myocyte (C). Myocytes with bizarre-shaped nuclei were frequently seen (D) (H&E stain; magnification: A–C ×20, D ×40).

Striking variations in the size of neighbouring myofibres were noted (fig 1C). Myocytes with bizarre-shaped nuclei were frequently encountered (fig 1D). There was scant, focal evidence for mild fibrosis (not shown).

Genome scan and mutation analysis

We hypothesised that a founder mutation was responsible for the severe infantile form of this HCM and therefore adopted a homozygosity mapping approach to identify the chromosomal location of the causative gene. Chromosomal regions were assayed using the datasets obtained from the first four affected subjects to identify common haplotypes. This approach identified a shared region of homozygosity on chromosome 11p11.2 encompassing the MYBPC3 gene, which is known to be associated with autosomal dominant forms of HCM. Analysis using microsatellite markers in this region confirmed autozygosity (data not shown).

Sequence analysis of coding exons and associated splice junctions of the MYBPC3 gene in these cases showed a splice site mutation in intron 30 (3330+2T>G) which was homozygous in all the affected children (fig 2). This mutation was not present in 100 control chromosomes of European ancestry from subjects younger than 60 years with no history of cardiovascular disease. However, these controls were not evaluated by echocardiography for subclinical cardiac disease. Computational spice site analysis using the spliceview program (http://zeus2.itb.cnr.it/∼webgene/wwwspliceview_ex.html, accessed 28 July 2008) indicates a predicted detrimental effect of the 3330+2T>G variant upon the probability of splicing occurring at the exon 30/intron 30 donor site.

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Figure 2 Sequencing results showing MYBPC3 gene variant.

DISCUSSION

We report the identification of a lethal infantile form of hypertrophic cardiomyopathy at high frequency in the Old Order Amish associated with homozygosity for the (3330+2T>G) splice site mutation. This mutation has recently been reported in three patients who presented with a similar aggressive infantile onset cardiomyopathy.11 This study represents a much larger clinical sample that illustrates the remarkably consistent presentation of the homozygous affected infants; the disorder has proved to be lethal in all cases that have not received orthotopic heart transplantation. All have respiratory distress, poor feeding and failure to thrive. While echocardiography on the first day of life may show preserved systolic function, follow-up studies uniformly demonstrate a progressive deterioration in systolic function. The ventricular dimensions were normal, which is not an unusual finding in children with HCM-causing mutations and diastolic dysfunction versus adults as left ventricular hypertrophy only occurs in a small percentage of children.1 Small muscular ventricular septal defects were common and showed bidirectional low-velocity shunting consistent with increased pulmonary artery pressures and resistance. Arrhythmias were not noted during clinical deterioration in any of these infants and death occurred as the result of inadequate cardiac output. In contrast to some forms of HCM, the Amish infants did not have fixed or dynamic left ventricular outflow tract obstruction. Although evidence for the diagnosis of HCM is clear in most cases, reliance upon parental recollections in the absence of access to the original clinical reports leads to ambiguities in some cases. The severity of the phenotype and the retrospective nature of the current study prohibits the reinvestigation of affected hearts of such cases to confirm the original clinical findings and diagnosis.

The microscopic features are typical for HCM, which is characterised by myocardial disarray in at least 20% of tissue examined.16 In the case presented here, this criterion was easily fulfilled because the disarray was widespread and affected both left and right ventricles. In contrast to adult cases of HCM, these affected infants showed no evidence of ischaemic change with scarring, significant myocardial fibrosis or coronary artery abnormalities. Several of the affected infants underwent extensive clinical evaluation for metabolic disorders and the results of these investigations were consistently inconclusive. Medical management that included digoxin, diuretics, afterload reduction and β blockade had no impact on the progressive clinical deterioration experienced by these patients. The only infants to survive underwent the extreme treatment of orthotopic heart transplantation.

The sarcomeric cardiac myosin binding protein C plays important structural and regulatory roles within the sarcomere.17 While frequencies are population-specific, defects in MYBPC3 are estimated to be responsible for about 15% or more of all HCM cases.7 18 Although there is variability in disease onset and prognosis in general, several studies report association of autosomal dominant MYBPC3 mutations with later onset, low penetrance and better prognosis.12 19 Our genetic findings are consistent with our previous genealogical studies, which indicate that Amish patients homozygous for genetic mutations have descended from common ancestors, suggesting that this variant also represents a founder mutation.14 15

The 3330+2T>G mutation identified in the affected patients is located at the exon 30/intron 30 splice site. Our computational analysis of this variant suggests that it strongly reduces the probability of splicing occurring at this donor site. Unfortunately, mRNA was not available to elucidate fully the exact effect of the 3330+2T>G mutation upon the MYBPC3 transcript. However, the computational predictions suggest either the continued reading of exon 30 into intron 30 which contains an inframe stop codon 111 bp into the intron or potential skipping of the 140 bp exon 30 itself, which would disrupt the reading frame leading to premature termination. The premature termination of both these predicted transcript outcomes may result in non-sense mediated decay. However, it is interesting to note that a recent study has shown MYBPC3 mutations that create a premature termination codon but which do not result in non-sense-mediated mRNA decay and are associated with accelerated degradation by an impaired ubiquitin–proteasome system.20

Homozygous or compound heterozygous mutations in sarcomeric genes including MYBPC3 have only rarely been reported and are typically missense mutations which are associated with adult-onset disease.7 21^–23 Two cases of neonatal onset HCM associated with MYBPC3 mutations have been reported; both cases were found to be compound heterozygous for truncating mutations and both patients died within the first few weeks of life.24 Another study described a homozygous MYBPC3 mutation (Q76ter) associated with congestive heart failure resulting in death of the child at 9 months of age.7 Interestingly, the clinical features of all three cases closely resembled those of the Amish neonatal disease. The more aggressive phenotypic presentation of these cases and the Amish neonatal lethal cardiomyopathy presented here, may reflect a more detrimental nature of the mutations in these patients compared with the homozygous mutations identified in adult-onset disease.

This study presents an important description of the cardiac abnormalities associated with a homozygous splice site mutation (3330+2T>G) in the MYBPC3 gene in 10 affected cases. The severity of the presentation is clear; the only surviving patients who are homozygous for the identified mutation received orthotopic heart transplantation. Heterozygous mutations in MYBPC3 are often associated with adult-onset HCM; however, the scope of cardiac involvement in heterozygous carrier parents in the current study is currently unclear. The majority are asymptomatic in their twenties but a more thorough investigation of carrier parents is required to assess any potential risk to their cardiac function.