Download A Novel Splice Donor Site Mutation in the MYBPC3 Gene is

A Novel Splice Donor Site Mutation in the MYBPC3 Gene is
Associated with Severe Neonatal Hypertrophic Cardiomyopathy
Baozhong Xin1, Erik Puffenberger2, John Tumbush1, J.R. Bockoven3, Heng Wang1
1Das Deutsch Center (DDC) Clinic for Special Needs Children, Middlefield, OH; 2The Clinic for Special Children, Strasburg, PA;
3The Heart Center, Akron Children Hospital, Akron, OH
Abstract
Hypertrophic cardiomyopathy is inherited in an autosomal dominant pattern with incomplete penetrance and variable clinical presentations. Mutations in the myosin-binding protein C (MYBPC3)
gene are one of the most frequent genetic causes of the disease. Patients with MYBPC3 mutations generally have a late onset and a good prognosis. We report here more than 20 Old Order Amish
children with severe neonatal hypertrophic cardiomyopathy caused by a novel homozygous splice site mutation in the MYBPC3 gene. The affected children typically presented with signs and
symptoms of congestive heart failure during the first three weeks of life. Echocardiography revealed hypertrophic non-obstructive cardiomyopathy. These children had a life span averaging 3 to 4
months. All patients died from heart failure before one year of age unless they received a heart transplant. A genome-wide autozygosity mapping study was performed in three patients using highdensity single nucleotide polymorphism (SNP) genotyping arrays. The disease related gene was localized to a 4.6 Mb region on chromosome 11p11.2-p11.12. This region contained 37 characterized
genes, including MYBPC3. Sequence analysis of the MYBPC3 gene in the probands identified a novel homozygous mutation, c.3330+2T>G, in the splice-donor site of intron 30, which resulted in
skipping of the 140-bp exon 30 and a frame shift with a premature stop codon in exon 31 (p.Asp1064GlyfsX38). We speculate a fairly high prevalence of this phenotype in Old Order Amish
communities and many individuals carrying a single c.3330+2T>G allele at risk for development of hypertrophic cardiomyopathy.
Introduction
Result
Hypertrophic cardiomyopathy (HCM) is inherited as an autosomal dominant disease
characterized by unexplained left ventricular hypertrophy. The prevalence of the condition is
as high as 1 in 500 in the general population. A prominent feature of HCM is the remarkable
clinical diversity, ranging from no symptoms to severe heart failure and sudden cardiac death.
Genetic causes of HCM are also fairly diverse with more than 400 mutations identified in at
least 9 genes encoding sarcomeric proteins.
Genotyping and mapping: A genome-wide autozygosity mapping localized the disease
gene to a 4.6 Mb interval on chromosome 11p11.2-p11.12 (Fig. 2).
Mutational analysis: A novel homozygous mutation in the splice donor site of intron 30 of
MYBPC3, c.3330+2T>G, was identified in all patients (Fig. 3).
Mutations in cardiac myosin-binding protein C (MYBPC3) gene are one of the most frequent
genetic causes of HCM. In general, patients with HCM caused by MYBPC3 mutations have a
more favorable clinical profile, characterized by a late onset and a relatively good prognosis.
Homozygous mutation in the MYBPC3 gene causing severe HCM has not been reported to
our knowledge. Here we describe a cohort of patients with severe HCM presenting in the
neonatal period, caused by homozygosity for a novel mutation in the MYBPC3 gene.
RNA analysis: RT-PCR and sequence analysis revealed skipping of the exon 30 in MYBPC3
transcript resulted from c.3330+2T>G variant (Fig. 4).
Methods
Discussion
Subjects: The 23 affected infants were Old Order Amish, with 21 from Ohio and 2 from
New York. HCM was clinically diagnosed and confirmed by echocardiography in each
patient. All patients died from heart failure before one year of age unless they received a
heart transplant. DNA samples from the patients, their parents and siblings were acquired
with informed consent.
Genotyping and mapping: High-density SNP genotyping arrays were used to map the
disease locus in three affected individuals from three families (Fig. 1).
Mutation analysis: PCR primers were designed to amplify each of the 34 protein-coding
exons and their flanking intronic sequences of MYBPC3. Direct sequencing was performed
on an Applied Biosystems 310 Genetic Analyzer
Homozygosity for the c.3330+2T>G mutation in the MYBPC3 gene is associated with a
severe neonatal HCM. The identification of a single mutation in different families implies a
founder effect for this severe condition.
In families with shared genetic heritage, genome-wide SNP arrays allow disease gene
mapping studies to be incorporated into routine diagnostic evaluations.
It remains concerning that many unidentified individuals in the community carrying a single
c.3330+2T>G allele might be at risk of developing HCM. There is an urgent need to develop
a practical strategy to deliver medical services to these individuals.
RNA Isolation and cDNA Amplification: Total RNA was isolated from whole blood.
The cDNA was synthesized and amplified using primers covering exons 29 through 31.
Fig. 3 - Identification of c.3330+2T>G mutation in the MYBPC3 gene. Partial sequence of
the boundary region of exon and intron 30 is shown and the three genotypes with respect to
c.3330+2T>G mutation are presented (arrows). All patients were homozygous for the
mutation, their parents were heterozygous, and no unaffected siblings were homozygous for
the change.
Fig.1 - Pedigrees of the 3 families used in the mapping study and mutational analysis of
hypertrophic cardiomyopathy. The probands are indicated with an arrow in each family.
Fig. 4 - Detection of the aberrant transcript as a consequence of c.3330+2T>G mutation.
RT-PCR amplification of MYBPC3 cDNA prepared from two heterozygous carriers (C)
yielded two products: the expected 367-bp fragment and an abnormal shorter product. Direct
sequencing of the PCR products revealed skipping of the 140-bp exon 30 in the shorter
fragment which led to a frame shift and premature stop codon in exon 31
(p.Asp1064GlyfsX38). As a control, RT-PCR products from two homozygous normal
individuals (N) only gave rise to the expected 367-bp product. M, DNA molecular marker.
Fig.2 - Autozygosity mapping of HCM gene in three affected individuals. A large, shared
block of homozygous SNPs was identified on chromosome 11p11.2-p11.12. The Location
scores were maximized in this region providing the greatest relative likelihood for gene
localization to this 4.6 Mb region. A known HCM-related gene, MYBPC3, was located in this
shared region and selected for mutation analysis.
Acknowledgements
We thank the Amish families in this report for their support. The study was supported in part by The Elisabeth Severance Prentiss Foundation.