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Hypertrophic cardiomyopathy: two homozygous cases with “typical” hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy
BBRC Biochemical and Biophysical Research Communications 309 (2003) 391–398 www.elsevier.com/locate/ybbrc
Hypertrophic cardiomyopathy: two homozygous cases with “typical” hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy Luisa Nanni,a,d Maurizio Pieroni,b,d Cristina Chimenti,b,d Barbara Simionati,a Rosanna Zimbello,a Attilio Maseri,c,d Andrea Frustaci,b and Gerolamo Lanfranchia,d,* a
CRIBI Biotechnology Center, Universit a degli Studi di Padova, Padua, Italy Cardiology Department, Universit a Cattolica del Sacro Cuore, Rome, Italy c Cardio-Thoracic and Vascular Department, Universit a Vita-Salute San Raffaele, Milan, Italy Center for Post-genomic Cardiovascular Research, Universita Vita-Salute San Raffaele, Milan, Italy b
d
Received 4 August 2003
Abstract About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed “typical” HCM (group B). Mutational analysis of the b-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head–rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially “malignant” defects as for the associated phenotypes, but no specific mutations of HCM/DCM. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Hypertrophic cardiomyopathy; Dilated cardiomyopathy; Mutation detection; Myosin heavy chain 7; Myosin-binding protein C; Troponin T2; Homozygous mutation; Compound heterozygous
Hypertrophic cardiomyopathy (HCM; MIM# 192600) is clinically defined by the presence of a hypertrophic, nondilated left ventricle (LV) in the absence of an increased external load, and pathologically by myocyte hypertrophy, disarray, and interstitial fibrosis. Left ventricular global systolic function, as indicated by the ejection fraction, is preserved and it can often appear supra normal, while the impaired diastolic relaxation leads to an increased end-diastolic pressure and symptoms of heart failure [19,31]. HCM affects one in 500 individuals according to echocardiographic criteria [18]. The clinical outcomes of HCM are highly variable and range from asymptomatic benign course to heart failure or sudden cardiac death (SCD). * Corresponding author. Fax: +39-049-827-6280. E-mail address: [email protected] (G. Lanfranchi).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.08.014
HCM is inherited as an autosomal dominant trait; 2/3 of the patients has a family history of HCM or SCD, while in the remainder of sporadic cases the causingmutation arises de novo. The genes encoding b-myosin heavy chain (MYH7; MIM# 160760) [9], myosin-binding protein C (MYBPC3; MIM# 600958) [2], and cardiac troponin-T (TNNT2; MIM# 191045) [34] are the most common genes involved, accounting for approximately 3/4 of all HCM cases, with mutations acting through a dominant-negative mechanism [17]. To date almost 150 causative mutations in 10 sarcomeric genes have been identified (see www.angis.org.au/Databases/ Heart/heartbreak.html), and the clinical variability featuring HCM may be partly due to the genetic substrate [28]. Approximately 10% of HCM cases progress to LV dilatation resembling dilated cardiomyopathy (DCM),
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that is characterized by impaired LV function, wall thinning, and cavity dilatation, with worsened signs and symptoms of congestive heart failure, combining systolic and diastolic dysfunction [32]. The macro- and microscopic morphology of the ventricle, the vascular structure and function, the age at the onset of HCM [3], and isolated mutations in MYH7, MYBPC3, TNNT2, and a-tropomyosin (TPM1; MIM# 191010) genes have been implicated in this unfavorable evolution [7,15,16,26], but its genesis remains to be clarified. The myocellular calcium dysregulation may be a primary trigger of DCM and heart failure as indicated by the recently reported mutations in the phospholamban gene (PLN; MIM# 172405). This gene codes for a transmembrane phosphoprotein that inhibits the cardiac sarcoplasmic reticular Ca2þ -ATPase (SERCA2a) pump. A missense mutation and null PLN gene have been associated with inherited DCM and heart failure cases [11,29], while a mutation of PLN promoter has been reported in a pt with familial HCM [21]. In order to investigate whether specific mutations of selected HCM genes are consistently associated with progression to DCM, we have performed mutational analysis of MYH7, MYBPC3, and TNNT2 in 11 unrelated affected patients (pts) progressed to DCM (group A) and 11 unrelated pts affected with “typical” HCM (group B).
Materials and methods Clinical evaluation. After informed pt consent and approval by the Ethics Committee of our institution, blood was drawn from 22 unrelated individuals seen at the A. Gemelli University Hospital, Rome, and diagnosed with HCM. Eleven pts (mean age 48.2 18.3 years, range 20–70 years) presented HCM progressed to the dilated phase by diagnostic findings before and after a mean follow-up period of 4 years and 5 months (group A). Five out of 11 reported a positive family history of HCM or SCD in a first-degree relative. All pts were symptomatic with exertional dyspnea, palpitation, and/or chest pain, and one pt with ventricular fibrillation was treated with an implantable cardioverter–defibrillator (ICD). A second group of pts included 11 unrelated individuals (mean age 45.3 11.3 years, range 27–61 years) diagnosed with “typical” HCM with preserved systolic function (group B). Three of them reported a positive family history. The clinical outcome in this group ranged from asymptomatic course to exertional dyspnea. All pts underwent cardiac catheterization with coronary and LV angiography as well as biventricular endomyocardial biopsy to rule out an underlying coronary artery disease and to confirm the diagnosis of HCM. Mutational analysis. Genomic DNA was isolated from peripheral blood lymphocytes using the QIAmp DNA Blood Mini kit from Qiagen. In the 22 enrolled pts, we have analyzed the coding regions and the exon–intron boundaries of MYH7, MYBPC3, and TNNT2 genes. We have obtained 16 amplicons for MYH7, 15 amplicons for MYBPC3, and seven amplicons for TNNT2, using two previously published primers (available at http://genetics.med.harvard.edu/~seidman.html) and 74 primers designed in our laboratory, according to the gene deposited sequences (GenBank Accession Nos. MYH7, M57965; MYBPC3, U91629; and TNNT2, AY044273). Primer sequences are
available on request (from the authors). For each pt, PCR amplification of the amplicons was performed in a 25 ll reaction volume (Tris– HCl 65.5 mM, (NH4 )2 SO4 16 mM, MgCl2 1.5 mM, and Tween 20, 0.01%), using 30 ng DNA template, 200 lM dNTPs, 16 pmol of each primer, and 1 U Taq polymerase. PCR conditions were as follows: 94 °C for 5 min, 32 cycles at 94 °C for 40 s, 55–59 °C for 30 s, and 72 °C for 40 s, followed by a final elongation step at 72 °C for 6 min. Amplified products were treated with exonuclease I and shrimp alkaline phosphatase enzymes (5 and 0.5 U, respectively, USB) to inactivate unused primers and dNTPs, sequenced by dye terminator cyclesequencing, and analyzed using a 96-capillary automated DNA sequencer (Applied Biosystem). Sequence variations in MYH7 and MYBPC3 that alter restriction enzyme sites were confirmed by PCR-amplification of relevant exons, digestion with the appropriate restriction enzymes, and analysis on 2% agarose gels, as reported in Results. The remaining sequence variants were confirmed by sequencing the opposite strand from independent PCR products. The DNAs of 100 unrelated normal Caucasian individuals were used as controls: three variants were tested by direct sequencing and two by the amplification refractory mutation system (ARMS) [22].
Results Table 1 summarizes the clinical profiles of the 22 individuals affected by HCM who have been enrolled in this study. In group A-pts, hypertrophy was mainly confined to the interventricular septum (IVS) and only pt 1A showed apical hypertrophy. LV angiography showed LV dilatation with reduced contractility in all A-pts, over a mean follow-up period of 4 years and 5 months from the diagnosis of HCM. The average left ventricular end-diastolic diameter (LVEDD) varied from 42.3 2.1 mm at diagnosis to 48.2 3.6 mm at follow-up. The mean left ventricular ejection fraction (LVEF) was 63.9 3% at diagnosis and decreased to 44.5 11.1% at follow-up. In no pt of group A, an outflow or intraventricular pressure gradient was measured. Group B-pts were characterized by an asymmetric hypertrophy, mainly of moderate degree (mean IVS 22.3 4.7 mm), but two of them (3B and 4B) showed a severe LV hypertrophy (32 and 27 mm). Echocardiographic and/or angiographic parameters at follow-up did not show a progression to the dilated phase in group B-pts (data not shown). A resting LV outflow tract gradient (LVOTO) was found in 4/11 pts (average 50 8.2 mmHg), but none of them presented a systolic dysfunction (mean LVEF 65.3 3.5%). Endomyocardial biopsy showed hypertrophic myocardiocytes with marked disarray, short runs, and perinuclear halo, confirming the diagnosis of HCM in all pts. Coronary arteries were normal in all pts as judged by coronary angiography. We performed a mutational analysis of the coding regions and exon–intron boundaries of the MYH7, MYBPC3, and TNNT2 genes in 11 pts presented with HCM progressed to DCM and 11 pts with typical HCM. No relevant variations were found in the TNNT2 sequence in all subjects, except for the presence of few
Table 1 Clinical profiles of HCM patients Age at Dg/Sex
Age at study
IVS (mm)
LVEDD (mm)
LVEF (%)
II
I
II
I
II
1A 2A 3A 4A 5A 6A 7A 8A 9A 10A 11A
20/F 33/F 20/F 63/M 57/M 40/M 51/M 34/M 60/F 64/M 39/F
20 36 23 70 63 44 55 37 69 69 44
a
a
25 15 18 17 20 17 25 22 19 21
25 10 14 14 16 13 23 16 10 18
45 40 44 41 41 45 43 39 43 44 40
47 43 50 47 52 49 51 44 51 53 43
70 65 60 65 65 63 66 60 64 60 65
67 60 50 45 45 41 37 35 35 30 45
1B 2B 3B 4B 5B 6B 7B 8B 9B 10B
M M M M M M M F F M
45 38 39 27 56 29 52 56 61 53
15 24 32 27 20 25 18 21 19 23
41 39 37 38 43 45 43 44 46 44
11B
M
42
21
45
I
LVOTO (mm Hg)
Symptoms
NYHA class
Family history
No No No No No No No No No No No
VF (ICD) Dyspnea, Palp Dyspnea, Palp Angina, dyspnea Angina, dyspnea Dyspnea Dyspnea Dyspnea Dyspnea, Palp Dyspnea Dyspnea
II III III III II II IV III III IV III
HCM HCM SCD No No No HCM No No No HCM
68 66 60 65 62 64 60 68 67 70
No 60 50 50 40 No No No No No
Mild dyspnea Angina, dyspnea Dyspnea NSVT at Holter Dyspnea, Palp Dyspnea Dyspnea Dyspnea Dyspnea Dyspnea
II II II II III III II II I I
No No HCM No No HCM No No No No
69
No
Dyspnea, Palp
II
HCM
Mutations
Group A MYH7-R453C MYH7-L517M
MYH7-Q734E
MYBPC3-Q1012X
Group B MYBPC3-E258K MYBPC3-R810Hb MYBPC3-P873Hb
MYBPC3-R810H; R820Q
Group A serial observations showing progression of HCM to DCM have been reported (I, II—clinical evaluation at different times). F, female; M, male; Dg, diagnosis; IVS, interventricular septum; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; LVOTO, left ventricular outflow tract obstruction; VF, ventricular fibrillation; ICD, implantable cardioverter–defibrillator; Palp, palpitations; NSVT, nonsustained ventricular tachycardia. a Apical and RV hypertrophy. b Homozygous mutation.
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Patient no.
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Table 2 Summary of sequence variations identified in the present study Gene
Sequence change
Expected effect
Confirmation Analysis
Reference
Pt. no.
MYH7a
g.10437C > T g.10913C > A g.13652C > G
R453C L517M Q734E
Rev. seq. Loss of AvaII; 200 neg. chr. (seq) Rev.seq; 200 neg. chr (seq)
[35] This paper This paper
2A 3A 8A
MYBPC3b
g.5256G > A g.16085G > A g.16115G > A g.17653C > A g.19935C > T
E258K R810H R820Q P873H Q1012X
Rev. seq. Rev.seq.; 200 neg.chr. (ARMS) Rev.seq.; 200 neg.chr. (seq.) Loss of BstXI; 200 neg.chr. (seq.) Loss of AlwNI; 200 neg.chr. (ARMS)
[23] This paper [15] This paper This paper
2B 3Bc ; 10B 10B 4Bc 11A
Rev. seq., reverse primer sequence; neg.chr., negative control chromosomes; ARMS: amplification refractory mutation system. Reference sequence, GenBank Accession No. M57965. b Reference sequence, GenBank Accession No. U91629. c Homozygous mutation. a
putative polymorphisms of doubtful etiologic role (data not shown). Considering both phenotypes (A and B), eight mutations of MYH7 or MYBPC3 were identified in eight pts. Three of these mutations have been previously reported as HCM-mutations and five are reported for the first time in this study (Table 2). Blood samples from parents of the pts carrying the mutations were not available for the study. Thus, we can suppose that one of the five new mutations could be de novo since it was found in a “sporadic” pt with a negative family history at anamnesis (8A). None of the five detected sequence variants was found in more than 200 control chromosomes from unrelated normal Caucasian individuals. Mutations in group A-pts Four different mutations found in four HCM/DCM pts occurred in the MYH7 and the MYBPC3 genes (Table 1). The first, MYH7-R453C, already known as a “malignant” mutation associated to high risk of SCD [35], was observed in a woman with moderate hyper-
trophy (2A) and a positive family history. In the protein, the R453C variant is located at the end of the ATPbinding pocket, facing toward the putative actin-binding region of the molecule, thus influencing the actin–myosin interaction [25]. This is the first time that this variant is associated with the HCM/DCM phenotype. The three following mutations are formerly unreported: the MYH7-L517M was detected in a woman with exertional dyspnea and palpitations (NYHA class III symptoms), whose father succumbed to SCD (3A). The 10913C > A-sequence change occurs in the 50 kDa fragment of the S1 subunit, at an invariant position in the myosin proteins (Figs. 1 and 2). This mutation was confirmed by the loss of an AvaII restriction site (data not shown). The MYH7-Q734E is a de novo mutation observed in a man with moderate hypertrophy, who showed 35% of LVEF (8A). The 13652C > G-sequence change replaces an uncharged and conserved glutamine (Q) with a negatively charged glutamic acid (E), in the 20 kDa fragment of the S1 subunit (Figs. 1 and 2). The third novel mutation found is a nonsense mutation of MYBPC3 gene (Q1012X) detected in a woman with
Fig. 1. Structure of MYH7 and MYBPC3 proteins and location of selected HCM-mutations. The globular domain of MYH7 can be proteolyzed into S1-head (further subdivided into 25, 50, and 20 kDa fragments) containing a catalytic ATPase site and an actin-binding site, and S2-rod. The a-helical coiled-coil light meromyosin (LMM-tail) directs the assembly of myosin into thick filaments. MYBPC3 consists of repeated domains with homology to immunoglobulin C2 (ovals) or fibronectin type 3 (squares) domains. Cardiac MYBPC3 isoform contains an additional N-terminal domain (C0), three specific phosphorylation sites in the MYBPC motif, and a proline/charged residue-rich insertion into domain C5. The novel mutations identified in this study are included in a red box.
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Fig. 2. Species comparison of amino acid sequences flanking the eight mutant residues found in 8/22 pts. The predicted amino acid changes are shown. Conserved amino acids are shown in red. In MYH7, the reactive cysteine residues are in green.
exertional dyspnea (NYHA class III symptoms), 45% of LVEF, and whose mother was also affected (11A). The 19935C > T-sequence change implies a premature termination of the protein at the Ig-like C8 domain (Fig. 1). This mutation was confirmed by the loss of an AlwNI restriction site (data not shown) in the pt DNA. Mutations in group B-pts In the group of pts with typical HCM, we have identified 4 different mutations of the MYBPC3 gene, two of which were previously unreported and identified in two homozygous pts and in a compound heterozygous-pt. The first mutation, MYBPC3-E258K, already reported as HCM-causing variant [23], was detected in a man with moderate hypertrophy and LVOTO (2B). This mutation flanks the MYBPC motif (Fig. 1) that contains three cardiac isoform-specific phosphorylation sites and confers a functional role to MYBPC3 in modulating the cardiac contraction. MYBPC3 becomes phosphorylated during adrenergic stimulation of the myocardium, with a concomitant increase in systolic tension [8]. Thus, the E258K variant may impair the contractile force through a dominant-negative mechanism on the MYBPC3 phosphorylation, during adrenergic stimulation. Moreover, given the proximity of E258K to splice signals, it might also alter RNA splicing with a premature termination of the protein. The two following variants are reported for the first time in this study. The MYBPC3-R810H has been detected in two unrelated pts (3B and 10B), as homozy-
gous and heterozygous mutation, respectively. Pt 3B showed severe hypertrophy (IVS 32 mm) and a LVOTO of 50 mmHg. He had a positive family history and came from a closed community of a little town that may justify the homozygous mutation. Pt 10B instead showed a moderate non-obstructive hypertrophy. The 16085G > A sequence change occurs at an invariant position, in the fibronectin type-3 domain (C6) of the MYBPC3 protein, whose function is still unknown (Figs. 1 and 2). Interestingly, pt 10B also carries the already known MYBPC3-R820Q mutation [15], a missense mutation that results in a change of charge of the altered amino acid, and occurs in the C6 motif in a conserved position (Table 2). Finally, we found the homozygous mutation MYBPC3-P873H in a man from a gipsy family (4B), in which the consanguinity between parents is very likely. This pt had a moderate/severe hypertrophy with LVOTO and showed a non-sustained ventricular tachycardia at Holter monitoring. The 17653C > A change involves a highly conserved residue in the fibronectin type-3 domain (C7), which is required for the proper integration of MYBPC3 into the thick filament (C-zones in A band of sarcomere) (Fig. 1). This mutation was confirmed by the loss of a BstXI restriction site (data not shown).
Discussion Our study shows that no recurrent mutations in MYH7, MYBPC3, and TNNT2 genes are consistently
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associated to HCM progressing to DCM, and are consistent with the heterogeneity of the disease. In fact, other genes besides the sarcomeric and cytoskeletal genes could also be involved in the onset of DCM, as suggested by the recent report on the association of PLN gene mutations with rare inherited cases of DCM and heart failure [11,21,29]. This justifies the low frequency of each mutation that seems exclusively associated to the individual or family in which it has been detected. Of the three novel HCM/DCM-associated mutations, two occurred in the MYH7 gene and the third in the MYBPC3 gene. To date only few mutations of sarcomeric genes and mitochondrial DNA have been reported in isolated HCM/DCM cases [7,15,16,26,33]. All the reported mutations can be defined as “maliglignant,” according to their location in the protein and the severity of the associated phenotype. The MYH7L517M variant locates close to the helix connecting the two reactive cysteine residues required for the ATPase activity (Cys-695 and Cys-705). Mutations located at this level might alter the nature and the timing of the conformational changes during the contractile cycle [25]. The MYH7-Q734E mutation is closely associated with structural elements that form the interface to the essential myosin light chain (MYL3; MIM# 160790) at the head–rod junction (Fig. 1). Since some MYL3 mutations lie in a region between MYL3 and MYH7 [24], and some MYH7 mutations, including Q734E, are spatially adjacent to these MYL3 mutations, it seems that the interface between heavy and light chains might be important for mechano-chemical coupling during contraction [25]. Of course, functional studies on the enzymatic and mechanical properties of these mutations might strengthen this hypothesis. Moreover, in the native structure of the protein, the residue Q-734 is closely adjacent to a positively charged residue lying in the putative actin-binding region (K-365). In the Q734E variant, a negatively charged residue (E) replaces the uncharged residue Q, thus leading to a stronger interaction between E734()) and K365(+) that may affect the actin–myosin binding. The third mutation detected in HCM pts progressing to DCM affects the MYBPC3 gene (Q1012X). Despite the overall benign nature of the MYBPC3 mutations [4,23], this nonsense mutation was detected in a pt with systolic dysfunction, suggesting that MYBPC3 mutations could sometimes cause a “malignant” phenotype [28]. In particular, the most severe manifestations of the disease have been associated to mutations leading to protein truncation [5]. The mechanisms by which MYBPC3 mutations lead to HCM are mostly unknown. In the A-band of the sarcomere, the last 102 amino acids bind the rod and tail of MYH7. MYBPC protein also interacts with titin (TTN; MIM# 188840), a giant protein that spans the length of the sarcomere
at multiple and regular intervals, depending on the three carboxy-terminal domains [6] (Fig. 1). The bindings of MYBPC3 to MYH7 and TTN confer stability to sarcomere organization. The Q1012X mutation leads to a protein truncation at the titin-binding site (C8). Conflicting results have been obtained about the mechanism by which the MYBPC3 protein lacking myosin and titin binding sites leads to HCM [10,30,36]. Functional studies of this mutation will be essential to elucidate if Q1012X acts through a dominant-negative mechanism, if the resulting protein continues to be incorporated into the A-band, or through haploinsufficiency, if the enhanced proteolysis of the truncated protein rather alters the stoichiometry of sarcomeric proteins. From analysis of pts with typical HCM, we identified two homozygous pts and a compound heterozygous pt of the MYBPC3 gene. The P873H variant was detected in a pt with severe and obstructive hypertrophy (4B). This mutation leads to the substitution of the non-polar residues (Pro) with positively charged residues (His) and occurs in the C7 domain of the protein that could interfere with the protein incorporation in the A-band of the sarcomere (Fig. 1). To date few cases of homozygous mutations associated to HCM are known, affecting the MYH7, TNNT2 or MYBPC3 gene [12,27,28], causing a severe cardiac phenotype. In a recent transgenic model of MYBPC3-related HCM, a deletion has been introduced via homologous recombination. Interestingly, the heterozygous mice showed a typical HCM, while the homozygous offspring developed a DCM [20]. These findings could be of clinical relevance given that, as in our group A-pts, about 10% of all HCM pts ultimately develop a DCM. Thus, pt 4B, clinically evaluated at the age of 27 years, might be part of that 10% of HCM pts and may require a specific attention at follow-up as for the possibility to develop DCM. He might otherwise represent a carrier of a homozygous mutation of MYBPC3 where a gene dosage effect occurs. The hypothesis of a gene dosage effect for MYBPC3 mutations is even more evident in the second group Bmutation. In fact, the MYBPC3-R810H variant was detected in heterozygosis in a 53-year-old man with moderate hypertrophy without obstruction (10B). This mutation was also found, though in homozygosis, in a 39-year-old pt with severe hypertrophy, LVOTO, and a positive family history (3B). The clinical pictures of 10B, 3B, and possibly 4B support a gene dosage effect for mutations in the MYBPC3 gene, where a homozygous mutation is associated with a more severe phenotype than the heterozygous. Of the 22 pts evaluated, those who carried a homozygous mutation showed a severe hypertrophy (3B and 4B). Pt 10B is a compound heterozygous with two MYBPC3 variants: R810H and R820Q. Both sequence
L. Nanni et al. / Biochemical and Biophysical Research Communications 309 (2003) 391–398
changes occur at an invariant position in the MYBPC3 proteins (Fig. 2) and were not found by sequencing 200 normal control chromosomes. The already known R820Q mutation has been associated with variable clinical features and the clinical expression of this mutation is often delayed until middle age, with a phenotype of HCM evolved to DCM in the 40% of the elderly carriers (age >70 years) [15]. Our pt was 53 years old at study and his compound heterozygosis may have contributed to the clinical expression of typical HCM, which may evolve into DCM with time. Few cases have been reported of compound/double heterozygous mutations in pts with HCM [1,13,14,28]. In all cases, the HCM phenotype was more severe when the two genetic variants were present simultaneously. In summary, the nature of the mutations, their location in the proteins plus the negative check in 200 normal chromosomes, and the conservation of the mutated residues among species make the five novel mutations etiologically relevant for HCM. The action of multiple gene mutations, modifier genes, genes involved in the myocellular calcium handling as PLN, and post-transcriptional mechanisms may play a major role in the progression of HCM toward DCM.
Acknowledgments This work was supported by the Ministero dell’ Universita e della Ricerca Scientifica, Italy (Grant COFIN 2002 No. 2002065411 to A.M. and G.L.), by the Istituto Superiore della Sanita, Italy (Progetto finalizzato No. 77/1999 to A.F. and G.L.), and by the Fondazione Internazionale di Ricerca per il Cuore Onlus, Rome.
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[31] J.G. Seidman, C. Seidman, The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms, Cell 104 (2001) 557–567. [32] P. Spirito, B.J. Maron, R.O. Bonow, S.E. Epstein, Occurrence and significance of progressive left ventricular wall thinning and relative cavity dilatation in hypertrophic cardiomyopathy, Am. J. Cardiol. 60 (1987) 123–129. [33] F. Terasaki, M. Tanaka, K. Kawamura, et al., A case of cardiomyopathy showing progression from the hypertrophic to the dilated form: association of Mt8348A ! G mutation in the mitochondrial tRNA(Lys) gene with severe ultrastructural alterations of mitochondria in cardiomyocytes, Jpn. Circ. J. 65 (2001) 691–694. [34] L. Thierfelder, H. Watkins, C. MacRae, et al., Alpha-tropomyosin and cardiac troponin T mutations cause familial hypertrophic cardiomyopathy: a disease of the sarcomere, Cell 77 (1994) 701– 712. [35] H. Watkins, A. Rosenzweig, D.S. Hwang, et al., Characteristics and prognostic implications of myosin missense mutations in familial hypertrophic cardiomyopathy, N. Engl. J. Med. 326 (1992) 1108–1114. [36] Q. Yang, A. Sanbe, H. Osinska, et al., A mouse model of myosin binding protein C human familial hypertrophic cardiomyopathy, J. Clin. Invest. 102 (1998) 1292–1300.
Hypertrophic cardiomyopathy: two homozygous cases with “typical” hypertrophic cardiomyopathy and three new mutations in cases with progression to dilated cardiomyopathy Luisa Nanni,a,d Maurizio Pieroni,b,d Cristina Chimenti,b,d Barbara Simionati,a Rosanna Zimbello,a Attilio Maseri,c,d Andrea Frustaci,b and Gerolamo Lanfranchia,d,* a
CRIBI Biotechnology Center, Universit a degli Studi di Padova, Padua, Italy Cardiology Department, Universit a Cattolica del Sacro Cuore, Rome, Italy c Cardio-Thoracic and Vascular Department, Universit a Vita-Salute San Raffaele, Milan, Italy Center for Post-genomic Cardiovascular Research, Universita Vita-Salute San Raffaele, Milan, Italy b
d
Received 4 August 2003
Abstract About 10% of cases of hypertrophic cardiomyopathy (HCM) evolve into dilated cardiomyopathy (DCM) with unknown causes. We studied 11 unrelated patients (pts) with HCM who progressed to DCM (group A) and 11 who showed “typical” HCM (group B). Mutational analysis of the b-myosin heavy chain (MYH7), myosin-binding protein C (MYBPC3), and cardiac troponin T (TNNT2) genes demonstrated eight mutations affecting MYH7 or MYBPC3 gene, five of which were new mutations. In group A-pts, the first new mutation occurred in the myosin head–rod junction and the second occurred in the light chain-binding site. The third new mutation leads to a MYBPC3 lacking titin and myosin binding sites. In group B, two pts with severe HCM carried two homozygous MYBPC3 mutations and one with moderate hypertrophy was a compound heterozygous for MYBPC3 gene. We identified five unreported mutations, potentially “malignant” defects as for the associated phenotypes, but no specific mutations of HCM/DCM. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Hypertrophic cardiomyopathy; Dilated cardiomyopathy; Mutation detection; Myosin heavy chain 7; Myosin-binding protein C; Troponin T2; Homozygous mutation; Compound heterozygous
Hypertrophic cardiomyopathy (HCM; MIM# 192600) is clinically defined by the presence of a hypertrophic, nondilated left ventricle (LV) in the absence of an increased external load, and pathologically by myocyte hypertrophy, disarray, and interstitial fibrosis. Left ventricular global systolic function, as indicated by the ejection fraction, is preserved and it can often appear supra normal, while the impaired diastolic relaxation leads to an increased end-diastolic pressure and symptoms of heart failure [19,31]. HCM affects one in 500 individuals according to echocardiographic criteria [18]. The clinical outcomes of HCM are highly variable and range from asymptomatic benign course to heart failure or sudden cardiac death (SCD). * Corresponding author. Fax: +39-049-827-6280. E-mail address: [email protected] (G. Lanfranchi).
0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.08.014
HCM is inherited as an autosomal dominant trait; 2/3 of the patients has a family history of HCM or SCD, while in the remainder of sporadic cases the causingmutation arises de novo. The genes encoding b-myosin heavy chain (MYH7; MIM# 160760) [9], myosin-binding protein C (MYBPC3; MIM# 600958) [2], and cardiac troponin-T (TNNT2; MIM# 191045) [34] are the most common genes involved, accounting for approximately 3/4 of all HCM cases, with mutations acting through a dominant-negative mechanism [17]. To date almost 150 causative mutations in 10 sarcomeric genes have been identified (see www.angis.org.au/Databases/ Heart/heartbreak.html), and the clinical variability featuring HCM may be partly due to the genetic substrate [28]. Approximately 10% of HCM cases progress to LV dilatation resembling dilated cardiomyopathy (DCM),
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that is characterized by impaired LV function, wall thinning, and cavity dilatation, with worsened signs and symptoms of congestive heart failure, combining systolic and diastolic dysfunction [32]. The macro- and microscopic morphology of the ventricle, the vascular structure and function, the age at the onset of HCM [3], and isolated mutations in MYH7, MYBPC3, TNNT2, and a-tropomyosin (TPM1; MIM# 191010) genes have been implicated in this unfavorable evolution [7,15,16,26], but its genesis remains to be clarified. The myocellular calcium dysregulation may be a primary trigger of DCM and heart failure as indicated by the recently reported mutations in the phospholamban gene (PLN; MIM# 172405). This gene codes for a transmembrane phosphoprotein that inhibits the cardiac sarcoplasmic reticular Ca2þ -ATPase (SERCA2a) pump. A missense mutation and null PLN gene have been associated with inherited DCM and heart failure cases [11,29], while a mutation of PLN promoter has been reported in a pt with familial HCM [21]. In order to investigate whether specific mutations of selected HCM genes are consistently associated with progression to DCM, we have performed mutational analysis of MYH7, MYBPC3, and TNNT2 in 11 unrelated affected patients (pts) progressed to DCM (group A) and 11 unrelated pts affected with “typical” HCM (group B).
Materials and methods Clinical evaluation. After informed pt consent and approval by the Ethics Committee of our institution, blood was drawn from 22 unrelated individuals seen at the A. Gemelli University Hospital, Rome, and diagnosed with HCM. Eleven pts (mean age 48.2 18.3 years, range 20–70 years) presented HCM progressed to the dilated phase by diagnostic findings before and after a mean follow-up period of 4 years and 5 months (group A). Five out of 11 reported a positive family history of HCM or SCD in a first-degree relative. All pts were symptomatic with exertional dyspnea, palpitation, and/or chest pain, and one pt with ventricular fibrillation was treated with an implantable cardioverter–defibrillator (ICD). A second group of pts included 11 unrelated individuals (mean age 45.3 11.3 years, range 27–61 years) diagnosed with “typical” HCM with preserved systolic function (group B). Three of them reported a positive family history. The clinical outcome in this group ranged from asymptomatic course to exertional dyspnea. All pts underwent cardiac catheterization with coronary and LV angiography as well as biventricular endomyocardial biopsy to rule out an underlying coronary artery disease and to confirm the diagnosis of HCM. Mutational analysis. Genomic DNA was isolated from peripheral blood lymphocytes using the QIAmp DNA Blood Mini kit from Qiagen. In the 22 enrolled pts, we have analyzed the coding regions and the exon–intron boundaries of MYH7, MYBPC3, and TNNT2 genes. We have obtained 16 amplicons for MYH7, 15 amplicons for MYBPC3, and seven amplicons for TNNT2, using two previously published primers (available at http://genetics.med.harvard.edu/~seidman.html) and 74 primers designed in our laboratory, according to the gene deposited sequences (GenBank Accession Nos. MYH7, M57965; MYBPC3, U91629; and TNNT2, AY044273). Primer sequences are
available on request (from the authors). For each pt, PCR amplification of the amplicons was performed in a 25 ll reaction volume (Tris– HCl 65.5 mM, (NH4 )2 SO4 16 mM, MgCl2 1.5 mM, and Tween 20, 0.01%), using 30 ng DNA template, 200 lM dNTPs, 16 pmol of each primer, and 1 U Taq polymerase. PCR conditions were as follows: 94 °C for 5 min, 32 cycles at 94 °C for 40 s, 55–59 °C for 30 s, and 72 °C for 40 s, followed by a final elongation step at 72 °C for 6 min. Amplified products were treated with exonuclease I and shrimp alkaline phosphatase enzymes (5 and 0.5 U, respectively, USB) to inactivate unused primers and dNTPs, sequenced by dye terminator cyclesequencing, and analyzed using a 96-capillary automated DNA sequencer (Applied Biosystem). Sequence variations in MYH7 and MYBPC3 that alter restriction enzyme sites were confirmed by PCR-amplification of relevant exons, digestion with the appropriate restriction enzymes, and analysis on 2% agarose gels, as reported in Results. The remaining sequence variants were confirmed by sequencing the opposite strand from independent PCR products. The DNAs of 100 unrelated normal Caucasian individuals were used as controls: three variants were tested by direct sequencing and two by the amplification refractory mutation system (ARMS) [22].
Results Table 1 summarizes the clinical profiles of the 22 individuals affected by HCM who have been enrolled in this study. In group A-pts, hypertrophy was mainly confined to the interventricular septum (IVS) and only pt 1A showed apical hypertrophy. LV angiography showed LV dilatation with reduced contractility in all A-pts, over a mean follow-up period of 4 years and 5 months from the diagnosis of HCM. The average left ventricular end-diastolic diameter (LVEDD) varied from 42.3 2.1 mm at diagnosis to 48.2 3.6 mm at follow-up. The mean left ventricular ejection fraction (LVEF) was 63.9 3% at diagnosis and decreased to 44.5 11.1% at follow-up. In no pt of group A, an outflow or intraventricular pressure gradient was measured. Group B-pts were characterized by an asymmetric hypertrophy, mainly of moderate degree (mean IVS 22.3 4.7 mm), but two of them (3B and 4B) showed a severe LV hypertrophy (32 and 27 mm). Echocardiographic and/or angiographic parameters at follow-up did not show a progression to the dilated phase in group B-pts (data not shown). A resting LV outflow tract gradient (LVOTO) was found in 4/11 pts (average 50 8.2 mmHg), but none of them presented a systolic dysfunction (mean LVEF 65.3 3.5%). Endomyocardial biopsy showed hypertrophic myocardiocytes with marked disarray, short runs, and perinuclear halo, confirming the diagnosis of HCM in all pts. Coronary arteries were normal in all pts as judged by coronary angiography. We performed a mutational analysis of the coding regions and exon–intron boundaries of the MYH7, MYBPC3, and TNNT2 genes in 11 pts presented with HCM progressed to DCM and 11 pts with typical HCM. No relevant variations were found in the TNNT2 sequence in all subjects, except for the presence of few
Table 1 Clinical profiles of HCM patients Age at Dg/Sex
Age at study
IVS (mm)
LVEDD (mm)
LVEF (%)
II
I
II
I
II
1A 2A 3A 4A 5A 6A 7A 8A 9A 10A 11A
20/F 33/F 20/F 63/M 57/M 40/M 51/M 34/M 60/F 64/M 39/F
20 36 23 70 63 44 55 37 69 69 44
a
a
25 15 18 17 20 17 25 22 19 21
25 10 14 14 16 13 23 16 10 18
45 40 44 41 41 45 43 39 43 44 40
47 43 50 47 52 49 51 44 51 53 43
70 65 60 65 65 63 66 60 64 60 65
67 60 50 45 45 41 37 35 35 30 45
1B 2B 3B 4B 5B 6B 7B 8B 9B 10B
M M M M M M M F F M
45 38 39 27 56 29 52 56 61 53
15 24 32 27 20 25 18 21 19 23
41 39 37 38 43 45 43 44 46 44
11B
M
42
21
45
I
LVOTO (mm Hg)
Symptoms
NYHA class
Family history
No No No No No No No No No No No
VF (ICD) Dyspnea, Palp Dyspnea, Palp Angina, dyspnea Angina, dyspnea Dyspnea Dyspnea Dyspnea Dyspnea, Palp Dyspnea Dyspnea
II III III III II II IV III III IV III
HCM HCM SCD No No No HCM No No No HCM
68 66 60 65 62 64 60 68 67 70
No 60 50 50 40 No No No No No
Mild dyspnea Angina, dyspnea Dyspnea NSVT at Holter Dyspnea, Palp Dyspnea Dyspnea Dyspnea Dyspnea Dyspnea
II II II II III III II II I I
No No HCM No No HCM No No No No
69
No
Dyspnea, Palp
II
HCM
Mutations
Group A MYH7-R453C MYH7-L517M
MYH7-Q734E
MYBPC3-Q1012X
Group B MYBPC3-E258K MYBPC3-R810Hb MYBPC3-P873Hb
MYBPC3-R810H; R820Q
Group A serial observations showing progression of HCM to DCM have been reported (I, II—clinical evaluation at different times). F, female; M, male; Dg, diagnosis; IVS, interventricular septum; LVEDD, left ventricular end-diastolic diameter; LVEF, left ventricular ejection fraction; LVOTO, left ventricular outflow tract obstruction; VF, ventricular fibrillation; ICD, implantable cardioverter–defibrillator; Palp, palpitations; NSVT, nonsustained ventricular tachycardia. a Apical and RV hypertrophy. b Homozygous mutation.
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Patient no.
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Table 2 Summary of sequence variations identified in the present study Gene
Sequence change
Expected effect
Confirmation Analysis
Reference
Pt. no.
MYH7a
g.10437C > T g.10913C > A g.13652C > G
R453C L517M Q734E
Rev. seq. Loss of AvaII; 200 neg. chr. (seq) Rev.seq; 200 neg. chr (seq)
[35] This paper This paper
2A 3A 8A
MYBPC3b
g.5256G > A g.16085G > A g.16115G > A g.17653C > A g.19935C > T
E258K R810H R820Q P873H Q1012X
Rev. seq. Rev.seq.; 200 neg.chr. (ARMS) Rev.seq.; 200 neg.chr. (seq.) Loss of BstXI; 200 neg.chr. (seq.) Loss of AlwNI; 200 neg.chr. (ARMS)
[23] This paper [15] This paper This paper
2B 3Bc ; 10B 10B 4Bc 11A
Rev. seq., reverse primer sequence; neg.chr., negative control chromosomes; ARMS: amplification refractory mutation system. Reference sequence, GenBank Accession No. M57965. b Reference sequence, GenBank Accession No. U91629. c Homozygous mutation. a
putative polymorphisms of doubtful etiologic role (data not shown). Considering both phenotypes (A and B), eight mutations of MYH7 or MYBPC3 were identified in eight pts. Three of these mutations have been previously reported as HCM-mutations and five are reported for the first time in this study (Table 2). Blood samples from parents of the pts carrying the mutations were not available for the study. Thus, we can suppose that one of the five new mutations could be de novo since it was found in a “sporadic” pt with a negative family history at anamnesis (8A). None of the five detected sequence variants was found in more than 200 control chromosomes from unrelated normal Caucasian individuals. Mutations in group A-pts Four different mutations found in four HCM/DCM pts occurred in the MYH7 and the MYBPC3 genes (Table 1). The first, MYH7-R453C, already known as a “malignant” mutation associated to high risk of SCD [35], was observed in a woman with moderate hyper-
trophy (2A) and a positive family history. In the protein, the R453C variant is located at the end of the ATPbinding pocket, facing toward the putative actin-binding region of the molecule, thus influencing the actin–myosin interaction [25]. This is the first time that this variant is associated with the HCM/DCM phenotype. The three following mutations are formerly unreported: the MYH7-L517M was detected in a woman with exertional dyspnea and palpitations (NYHA class III symptoms), whose father succumbed to SCD (3A). The 10913C > A-sequence change occurs in the 50 kDa fragment of the S1 subunit, at an invariant position in the myosin proteins (Figs. 1 and 2). This mutation was confirmed by the loss of an AvaII restriction site (data not shown). The MYH7-Q734E is a de novo mutation observed in a man with moderate hypertrophy, who showed 35% of LVEF (8A). The 13652C > G-sequence change replaces an uncharged and conserved glutamine (Q) with a negatively charged glutamic acid (E), in the 20 kDa fragment of the S1 subunit (Figs. 1 and 2). The third novel mutation found is a nonsense mutation of MYBPC3 gene (Q1012X) detected in a woman with
Fig. 1. Structure of MYH7 and MYBPC3 proteins and location of selected HCM-mutations. The globular domain of MYH7 can be proteolyzed into S1-head (further subdivided into 25, 50, and 20 kDa fragments) containing a catalytic ATPase site and an actin-binding site, and S2-rod. The a-helical coiled-coil light meromyosin (LMM-tail) directs the assembly of myosin into thick filaments. MYBPC3 consists of repeated domains with homology to immunoglobulin C2 (ovals) or fibronectin type 3 (squares) domains. Cardiac MYBPC3 isoform contains an additional N-terminal domain (C0), three specific phosphorylation sites in the MYBPC motif, and a proline/charged residue-rich insertion into domain C5. The novel mutations identified in this study are included in a red box.
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Fig. 2. Species comparison of amino acid sequences flanking the eight mutant residues found in 8/22 pts. The predicted amino acid changes are shown. Conserved amino acids are shown in red. In MYH7, the reactive cysteine residues are in green.
exertional dyspnea (NYHA class III symptoms), 45% of LVEF, and whose mother was also affected (11A). The 19935C > T-sequence change implies a premature termination of the protein at the Ig-like C8 domain (Fig. 1). This mutation was confirmed by the loss of an AlwNI restriction site (data not shown) in the pt DNA. Mutations in group B-pts In the group of pts with typical HCM, we have identified 4 different mutations of the MYBPC3 gene, two of which were previously unreported and identified in two homozygous pts and in a compound heterozygous-pt. The first mutation, MYBPC3-E258K, already reported as HCM-causing variant [23], was detected in a man with moderate hypertrophy and LVOTO (2B). This mutation flanks the MYBPC motif (Fig. 1) that contains three cardiac isoform-specific phosphorylation sites and confers a functional role to MYBPC3 in modulating the cardiac contraction. MYBPC3 becomes phosphorylated during adrenergic stimulation of the myocardium, with a concomitant increase in systolic tension [8]. Thus, the E258K variant may impair the contractile force through a dominant-negative mechanism on the MYBPC3 phosphorylation, during adrenergic stimulation. Moreover, given the proximity of E258K to splice signals, it might also alter RNA splicing with a premature termination of the protein. The two following variants are reported for the first time in this study. The MYBPC3-R810H has been detected in two unrelated pts (3B and 10B), as homozy-
gous and heterozygous mutation, respectively. Pt 3B showed severe hypertrophy (IVS 32 mm) and a LVOTO of 50 mmHg. He had a positive family history and came from a closed community of a little town that may justify the homozygous mutation. Pt 10B instead showed a moderate non-obstructive hypertrophy. The 16085G > A sequence change occurs at an invariant position, in the fibronectin type-3 domain (C6) of the MYBPC3 protein, whose function is still unknown (Figs. 1 and 2). Interestingly, pt 10B also carries the already known MYBPC3-R820Q mutation [15], a missense mutation that results in a change of charge of the altered amino acid, and occurs in the C6 motif in a conserved position (Table 2). Finally, we found the homozygous mutation MYBPC3-P873H in a man from a gipsy family (4B), in which the consanguinity between parents is very likely. This pt had a moderate/severe hypertrophy with LVOTO and showed a non-sustained ventricular tachycardia at Holter monitoring. The 17653C > A change involves a highly conserved residue in the fibronectin type-3 domain (C7), which is required for the proper integration of MYBPC3 into the thick filament (C-zones in A band of sarcomere) (Fig. 1). This mutation was confirmed by the loss of a BstXI restriction site (data not shown).
Discussion Our study shows that no recurrent mutations in MYH7, MYBPC3, and TNNT2 genes are consistently
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associated to HCM progressing to DCM, and are consistent with the heterogeneity of the disease. In fact, other genes besides the sarcomeric and cytoskeletal genes could also be involved in the onset of DCM, as suggested by the recent report on the association of PLN gene mutations with rare inherited cases of DCM and heart failure [11,21,29]. This justifies the low frequency of each mutation that seems exclusively associated to the individual or family in which it has been detected. Of the three novel HCM/DCM-associated mutations, two occurred in the MYH7 gene and the third in the MYBPC3 gene. To date only few mutations of sarcomeric genes and mitochondrial DNA have been reported in isolated HCM/DCM cases [7,15,16,26,33]. All the reported mutations can be defined as “maliglignant,” according to their location in the protein and the severity of the associated phenotype. The MYH7L517M variant locates close to the helix connecting the two reactive cysteine residues required for the ATPase activity (Cys-695 and Cys-705). Mutations located at this level might alter the nature and the timing of the conformational changes during the contractile cycle [25]. The MYH7-Q734E mutation is closely associated with structural elements that form the interface to the essential myosin light chain (MYL3; MIM# 160790) at the head–rod junction (Fig. 1). Since some MYL3 mutations lie in a region between MYL3 and MYH7 [24], and some MYH7 mutations, including Q734E, are spatially adjacent to these MYL3 mutations, it seems that the interface between heavy and light chains might be important for mechano-chemical coupling during contraction [25]. Of course, functional studies on the enzymatic and mechanical properties of these mutations might strengthen this hypothesis. Moreover, in the native structure of the protein, the residue Q-734 is closely adjacent to a positively charged residue lying in the putative actin-binding region (K-365). In the Q734E variant, a negatively charged residue (E) replaces the uncharged residue Q, thus leading to a stronger interaction between E734()) and K365(+) that may affect the actin–myosin binding. The third mutation detected in HCM pts progressing to DCM affects the MYBPC3 gene (Q1012X). Despite the overall benign nature of the MYBPC3 mutations [4,23], this nonsense mutation was detected in a pt with systolic dysfunction, suggesting that MYBPC3 mutations could sometimes cause a “malignant” phenotype [28]. In particular, the most severe manifestations of the disease have been associated to mutations leading to protein truncation [5]. The mechanisms by which MYBPC3 mutations lead to HCM are mostly unknown. In the A-band of the sarcomere, the last 102 amino acids bind the rod and tail of MYH7. MYBPC protein also interacts with titin (TTN; MIM# 188840), a giant protein that spans the length of the sarcomere
at multiple and regular intervals, depending on the three carboxy-terminal domains [6] (Fig. 1). The bindings of MYBPC3 to MYH7 and TTN confer stability to sarcomere organization. The Q1012X mutation leads to a protein truncation at the titin-binding site (C8). Conflicting results have been obtained about the mechanism by which the MYBPC3 protein lacking myosin and titin binding sites leads to HCM [10,30,36]. Functional studies of this mutation will be essential to elucidate if Q1012X acts through a dominant-negative mechanism, if the resulting protein continues to be incorporated into the A-band, or through haploinsufficiency, if the enhanced proteolysis of the truncated protein rather alters the stoichiometry of sarcomeric proteins. From analysis of pts with typical HCM, we identified two homozygous pts and a compound heterozygous pt of the MYBPC3 gene. The P873H variant was detected in a pt with severe and obstructive hypertrophy (4B). This mutation leads to the substitution of the non-polar residues (Pro) with positively charged residues (His) and occurs in the C7 domain of the protein that could interfere with the protein incorporation in the A-band of the sarcomere (Fig. 1). To date few cases of homozygous mutations associated to HCM are known, affecting the MYH7, TNNT2 or MYBPC3 gene [12,27,28], causing a severe cardiac phenotype. In a recent transgenic model of MYBPC3-related HCM, a deletion has been introduced via homologous recombination. Interestingly, the heterozygous mice showed a typical HCM, while the homozygous offspring developed a DCM [20]. These findings could be of clinical relevance given that, as in our group A-pts, about 10% of all HCM pts ultimately develop a DCM. Thus, pt 4B, clinically evaluated at the age of 27 years, might be part of that 10% of HCM pts and may require a specific attention at follow-up as for the possibility to develop DCM. He might otherwise represent a carrier of a homozygous mutation of MYBPC3 where a gene dosage effect occurs. The hypothesis of a gene dosage effect for MYBPC3 mutations is even more evident in the second group Bmutation. In fact, the MYBPC3-R810H variant was detected in heterozygosis in a 53-year-old man with moderate hypertrophy without obstruction (10B). This mutation was also found, though in homozygosis, in a 39-year-old pt with severe hypertrophy, LVOTO, and a positive family history (3B). The clinical pictures of 10B, 3B, and possibly 4B support a gene dosage effect for mutations in the MYBPC3 gene, where a homozygous mutation is associated with a more severe phenotype than the heterozygous. Of the 22 pts evaluated, those who carried a homozygous mutation showed a severe hypertrophy (3B and 4B). Pt 10B is a compound heterozygous with two MYBPC3 variants: R810H and R820Q. Both sequence
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changes occur at an invariant position in the MYBPC3 proteins (Fig. 2) and were not found by sequencing 200 normal control chromosomes. The already known R820Q mutation has been associated with variable clinical features and the clinical expression of this mutation is often delayed until middle age, with a phenotype of HCM evolved to DCM in the 40% of the elderly carriers (age >70 years) [15]. Our pt was 53 years old at study and his compound heterozygosis may have contributed to the clinical expression of typical HCM, which may evolve into DCM with time. Few cases have been reported of compound/double heterozygous mutations in pts with HCM [1,13,14,28]. In all cases, the HCM phenotype was more severe when the two genetic variants were present simultaneously. In summary, the nature of the mutations, their location in the proteins plus the negative check in 200 normal chromosomes, and the conservation of the mutated residues among species make the five novel mutations etiologically relevant for HCM. The action of multiple gene mutations, modifier genes, genes involved in the myocellular calcium handling as PLN, and post-transcriptional mechanisms may play a major role in the progression of HCM toward DCM.
Acknowledgments This work was supported by the Ministero dell’ Universita e della Ricerca Scientifica, Italy (Grant COFIN 2002 No. 2002065411 to A.M. and G.L.), by the Istituto Superiore della Sanita, Italy (Progetto finalizzato No. 77/1999 to A.F. and G.L.), and by the Fondazione Internazionale di Ricerca per il Cuore Onlus, Rome.
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