Myozenin 2 Is a Novel Gene for Human Hypertrophic Cardiomyopathy
Hypertrophic cardiomyopathy (HCM) is a genetic disorder caused by mutations in sarcomeric proteins (excluding phenocopy). The causal genes in approximately one-third of the cases remain unknown. We identified a family comprised of 6 clinically affected members. The phenotype was characterized by early onset of symptoms, pronounced cardiac hypertrophy, and cardiac arrhythmias. We excluded MYH7, MYBPC3, TNNT2, and ACTC1 as the causal gene either by direct sequencing or by haplotype analysis. To map the putative candidate sarcomeric gene, we perforbold locus-specific haplotyping to detect cosegregation of the locus haplotype with the phenotype, followed by mutation screening. We genotyped 5 short-tandem-repeat markers that spanned a 4.4-centimorgan region on 4q26-q27 locus and encompassed myozenin 2 (MYOZ2), a Z-disk protein. The maximum logarithm of odds score was 2.03 (P=0.005). All affected members shared a common haplotype, implicating MYOZ2 as the causal gene. To detect the causal mutation, we sequenced all exons and exon–intron boundaries of MYOZ2 in 10 family members and identified a T→C missense mutation corresponding to S48P substitution, which cosegregated with inheritance of HCM (N=6). It was absent in 4 clinically normal family members and in 658 additional normal individuals. To determine frequency of the MYOZ2 mutations in HCM, we sequenced MYOZ2 in 516 HCM probands and detected another missense mutation (I246M). It was absent in 2 normal family members and 517 controls. Both mutations affect highly conserved amino acids. We conclude MYOZ2 is a novel causal gene for human HCM.
Hypertrophic cardiomyopathy (HCM) is a genetic disease characterized by unexplained cardiac hypertrophy, myocyte disarray, and interstitial fibrosis (reviewed previously1). It is a major cause of sudden cardiac death in the young and diastolic heart failure.1 Molecular genetic basis of HCM has been partially elucidated. More than a dozen genes, all encoding sarcomeric proteins (except for phenocopy) have been identified.2,3 We describe MYOZ2, encoding myozenin 2 (calsarcin 1), as a novel gene for human HCM.
Materials and Methods
The study protocol was approved by the Institutional Review Board and was in accordance with the Human Subjects’ Committee guidelines (University of Texas Health Science Center, Houston). We collected blood samples from the consenting individuals and extracted genomic DNA by a conventional method. The participants underwent detailed history taking, physical examination, 12-lead electrocardiography, and transthoracic echocardiography. Additional procedures were performed as clinically indicated. HCM was diagnosed based on the presence of unexplained left ventricular wall thickness of ≥13 mm on the echocardiogram.
We identified a black family (designated as family 109) comprising 6 affected individuals, including a pair of dizygotic twins (Figure 1). The index case presented at 12 years of age with symptoms of diastolic heart failure and episodes of syncope (supplemental Table I available in the online data supplement available at http://circres.ahajournals.org.). She had severe left ventricular hypertrophy with a septal thickness of 3.2 cm, atrial fibrillation, and runs of ventricular tachycardia. The clinical characteristics of the affected family members are shown in the supplemental Table I.
We screened the candidate genes based on their frequencies as causes of HCM and novelty.1 We excluded mutations in MYH7, MYBPC3, TNNC1, and TNNT2 by direct sequencing. We used a locus-specific haplotyping to screen the less common candidates TNNI3, TPM1, ACTC1, TTN, and MYOZ2. We genotyped 5 to 6 short-tandem-repeat DNA markers at approximately 1-centimorgan (cM) intervals for each locus using fluorescently labeled primers and PCR (Tables II and III in the online data supplement, available at http://circres.ahajournals.org). The PCR fragments were separated by capillary electrophoresis on an ABI 3130xl Genetic Analyzer and analyzed using the GeneMapper v3.7 (Applied Biosystems).
We excluded ACTC1 by showing lack of cosegregation. In contrast, all 6 affected members shared a common haplotype for the MYOZ2 locus on 4q26-q27, whereas 4 clinically normal family members did not (Figure 1 and supplemental Figure I). Two asymptomatic family members (II-4 and III-8) declined to participate. The findings strongly implicated MYOZ2 as the putative causal genes. The remaining genes were not analyzed further.
The maximum logarithm of odds (LOD) score was 2.03 at markers D4S2303 and D4S1573, the closest markers to MYOZ2, assuming a disease allele frequency of 0.001 and penetrance of 0.99 in a dominant disease model (Figure 2 and supplemental Table IV). The LOD score is equivalent to a probability value of 0.005 adjusted for multiple testing of markers. We amplified and sequenced all 6 exons and exon–intron boundaries of MYOZ2 in 10 family members using the Big Dye Terminator Reaction in an ABI 3130xl Genetic Analyzer (supplemental Table II). Each sequence was analyzed for the presence of variants and compared with the GenBank sequence (NC_000004). We identified a heterozygous T→C missense (S48P) mutation at nucleotide position 15 072 (Figure 2). The mutation was present in all 6 affected members and absent in 4 clinically normal family members (Figure 1). The dizygotic twin brothers with the S48P mutation exhibited different degrees of asymmetric septal hypertrophy, which could reflect the effects of modifier genes and environmental factors (supplemental Table I). The locus comprises 30 genes including 9 encoding hypothetical proteins. None encodes a sarcomeric protein other than MYOZ2 or a known protein for HCM phenocopy. Nevertheless, the possibility of linkage disequilibrium with the actual causal mutation cannot be excluded with certainty.
To exclude the possibility of a rare polymorphism, we designed a 5′ nuclease assay and screened 658 normal individuals (asymptomatic with normal ECGs and echocardiograms), including 253 blacks by allelic discrimination on an ABI PRISM 7900HT SDS. The S48P variant was absent in 1316 normal chromosomes. Comparison of MYOZ2 protein sequence across species identified the serine 48 as a completely conserved amino acid (Figure 2).
To determine the frequency of MYOZ2 mutation in HCM, we screened all exons and exon–intron boundaries of MYOZ2 in 516 probands by direct sequencing. We detected another heterozygous missense A→G mutation at nucleotide 50 278 in a white proband who had 2 deceased siblings with HCM. The mutation changed amino acid isoleucine 246, a conserved amino acid, to methionine (Figure 2). Two offspring of the proband (54 and 33 years of age) were asymptomatic and had normal physical examination, ECGs, and echocardiograms. They did not carry the mutation. The mutation was also absent in 517 normal individuals (405 whites).
Results and Discussion
We detected several synonymous and intronic variants in MYOZ2 gene, which are shown in supplemental Table IV.
Under certain circumstances, the haplotype-sharing approach, restricted to the candidate loci, could facilitate mapping of the candidate genes in small families possessing classes of proteins that are known to cause the phenotype. Accordingly, it is applicable to genetic studies of primary cardiomyopathies caused by mutations in sarcomeric, cytoskeletal, and desmosomal proteins, particularly in small families, in which the conventional genome-wide linkage mapping may not offer sufficient power to map the causal gene.
Studies are ongoing to delineate the molecular mechanism(s) by which MYOZ2 mutations cause HCM. Myozenins are Z-disk proteins exclusive to striated muscles. They bridge Ca+2/calmodulin-regulated protein phosphatase calcineurin to α-actinin.4 MYOZ2 is specific to cardiac and slow-skeletal muscle fibers.5 All affected family members had normal skeletal muscle strength on physical examination. However, possibility of subclinical abnormalities was not excluded. MYOZ2 is a negative regulator of calcineurin functions.5 Germline deletion of Myoz2 in mice leads to activation of calcineurin, nuclear localization of NFAT, expression of cardiac hypertrophic genes, and enhanced cardiac hypertrophic response.5 We speculate MYOZ2 mutations cause HCM by activating the calcineurin pathway. The mutations, however, are not located within the known binding domains for calcineurin between amino acids 217 and 240.4 Similarly, the mutations are outside the α-actinin–binding domain of MYOZ2 between amino acids 153 to 200. The mutations could affect calcineurin signaling and/or binding to α-actinin by changing the secondary structure of MYOZ2. Alternative possibilities include interactions with proteins and/or altered mechanosignaling through the Z-disk, which was implicated in the pathogenesis of HCM caused by T-cap telethonin (TCAP), a Z-disk protein.6
Identification of MYOZ2 mutations highlights the diversity of molecular genetics and pathogenesis of HCM. Whether treatment with calcineurin inhibitors could prevent, attenuate, or reverse the phenotype in the subset of HCM caused by MYOZ2 mutations remains to be tested. Calcineurin inhibitors have been shown to impart deleterious effects on cardiac structure and function in α-MyHC-403 mice.7 Thus, it may be necessary to define different subsets of HCM at genetic and molecular levels to deliver gene-specific treatment for patients with HCM.
In summary, we have identified 2 mutations in MYOZ2 in patients with HCM. The haplotype and the S48P mutation cosegregated with inheritance of HCM. We also identified a second mutation (I246M) in a proband with HCM. Mutations were absent in ethnically matched controls and in the Single-Nucleotide Polymorphism database. They involved highly conserved amino acids. We conclude MYOZ2 mutations are uncommon causes of HCM (1:250). Studies are necessary to delineate the molecular mechanisms involved.
Sources of Funding
This work was supported in part by National Heart, Lung, and Blood Institute grant R01-HL68884, a Clinician-Scientist Award in Translation Research from the Burroughs Wellcome Fund (no. 1005907), and the Greater Houston Community Foundation (TexGen).
Original received December 30, 2006; resubmission received February 6, 2007; revised resubmission received February 26, 2007; accepted February 28, 2007.
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