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Cardiovascular Research 2003 57(2):347-357; doi:10.1016/S0008-6363(02)00711-3
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Copyright © 2003, European Society of Cardiology

Outcome of clinical versus genetic family screening in hypertrophic cardiomyopathy with focus on cardiac β-myosin gene mutations

Ole Havndrupa,*, Henning Bundgaarda, Paal Skytt Andersenb, Lars Allan Larsenb, Jens Vuustb, Keld Kjeldsena and Michael Christiansenb

aDepartment of Medicine B, The Heart Centre, Rigshospitalet, Copenhagen University Hospital, Blegdamsvej 9, 2100 Copenhagen, Denmark
bDepartment of Clinical Biochemistry, Statens Serum Institut, Copenhagen, Denmark

havndrup{at}dadlnet.dk

* Corresponding author. Tel.: +45-354-52-343; fax: +45-353-83-186.

Received 2 July 2002; accepted 16 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Genotype-phenotype...
 5. Discussion
 6. Conclusion
 References
 
Objective: Familial hypertrophic cardiomyopathy (FHC) is caused by mutations in genes encoding cardiac sarcomere proteins. Although available, genetic analyses are generally not used clinically. In the present study, we evaluated the outcome of clinical vs. genetic screening of family members with specific focus on mutations in the cardiac β-myosin heavy chain (MYH7) gene. Methods: A consecutive cohort of 68 FHC probands and their families (395 persons) of Danish origin was evaluated including patient- and family histories, physical examinations, electrocardiogram and echocardiography. Mutation screening was performed by a combination of single strand conformation/heteroduplex analysis and direct sequencing. Results: Eight different MYH7 gene mutations were identified in nine (13%) families (96 persons). In eight (89%) of the families, major cardiac events had occurred. Myectomy or percutaneous septal alcohol ablation had been performed in a higher number of MYH7 probands i.e. in five of nine (56%) as compared to 10 of 59 (17%) (P<0.05) non-MYH7 mutation probands. Neither echocardiographic nor ECG findings were useful to distinguish MYH7 from non-MYH7 probands. Between adult MYH7 mutation-carriers (n=38) and their non-carrier relatives (n=39), low sensitivity and specificity of the clinical diagnostic criteria tested were observed and minor clinical diagnostic criteria alone were not useful for identification of mutation carriers. By genetic screening of relatives with no or only minor hypertrophy on echocardiography, i.e. a priori possible mutation-carriers normally recommended clinical follow-up—the diagnosis was excluded in 52 (83%) persons. In addition, six relatives with secondary hypertrophy were identified as non-carriers. Conclusion: Neither echocardiographic nor ECG findings were useful to distinguish MYH7 from non-MYH7 probands. Extension of screening to include genetic analyses offered a marked diagnostic advantage as compared to clinical screening alone in FHC families.

KEYWORDS Cardiomyopathy; Hypertrophy; Sequence (DNA/RNA/prot); Sudden death

This article is referred to in the Editorial by C. Hengstenberg et al. (pages 298–301) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Genotype-phenotype...
 5. Discussion
 6. Conclusion
 References
 
Familial hypertrophic cardiomyopathy (FHC) is an inherited, autosomal dominant cardiac disease with a phenotypic prevalence of 1 in 500 [1]. Mutations in the cardiac β-myosin heavy chain gene (MYH7 gene) together with mutations in the myosin binding protein C gene (MYBPC3 gene) are suggested to account for up to 50% of FHC cases [2,3]. Mutations in other genes encoding sarcomere proteins also cause FHC [4–8]. On this basis FHC has been suggested to be a disease of the sarcomere with the abnormal protein acting as a dominant negative, ‘poisonous’ peptide altering the force of contraction, secondarily leading to myocardial hypertrophy [9]. FHC associated with MYH7 gene mutations has generally been characterized by an early onset of disease [10] especially compared to patients with mutations in the MYPBC3 gene who tend to have less hypertrophy early in life and to be free from symptoms until mid-life [11]. However, phenotypic heterogeneity is not only related to mutations in different genes but also to different mutations within the same gene. Thus, some of the presently more than 50 identified MYH7 gene mutations show almost full penetrance at the age of 20 years and have been associated with a marked reduction in lifetime, whereas other MYH7 gene mutations have reduced penetrance with low mortality rates [5,12,13]. Furthermore, between and even within families, a specific mutation may give rise to markedly different phenotypes. It has been expected that the evolving knowledge of genotype–phenotype relations in FHC might offer diagnostic as well as prognostic information and prove valuable when deciding genetic diagnostic strategies based on the clinical findings. Clinical screening of relatives to patients with FHC—including family history, physical examinations, ECG and echocardiography has generally been recommended. However, as indicated, differences in phenotypic presentation and age related penetrance make clinical screening difficult—and call for screening follow-up for several years—rather than one conclusive examination. Taken together, a genetic diagnosis may offer clinical important information for patient management as well as for screening of relatives. In approximately 50% of FHC families, a disease causing mutation may be found [2]. Once a mutation is identified in a family, genetic screening of family members may unambiguously characterize family members as carriers—with a high risk for development of the disease—and non-carriers with a risk as in the background population. In the present study, we compared the outcome of thorough clinical screening vs. genetic screening in 68 consecutively included FHC probands and their relatives with specific focus on identification and characterization of patients and families with mutations in one of the most frequently FHC associated genes, i.e. the MYH7 gene.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Genotype-phenotype...
 5. Discussion
 6. Conclusion
 References
 
2.1 Clinical studies
Sixty-eight families (395 persons) of Danish origin were included consecutively and prospectively in the present study of FHC at Rigshospitalet, Copenhagen University Hospital, Denmark. The hospital serves as tertiary referral centre for patients with FHC as well as primary centre for an additional local area population. Probands were included regardless of the severity of disease or evidence of familial or sporadic occurrence of disease. The investigation conformed with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1977;35:2–3) and, after informed consent was obtained (Local Science Ethics Committee Copenhagen; protocol no. KF V92213) and at the time of inclusion, patient- and family histories were obtained and physical examinations, 12-lead ECG, two-dimensional- and M-mode-echocardiography were performed. All probands fulfilled conventional diagnostic criteria for hypertrophic cardiomyopathy [14,15]. The clinical diagnosis of FHC in relatives was established before the genetic analyses were performed, and was based on: (A) ECG criteria: pathological Q-waves or signs of left ventricular hypertrophy as proposed [14,16,17] and/or (B) echocardiography criteria: a maximal left ventricular wall thickness of at least 13 mm [14,16,17] in the absence of evidence of other specific heart muscle diseases [15]. For deceased family members, clinical records and family histories were obtained whenever available. The ECGs were scored according to the Romhild–Estes and Sokolow classifications [18,19] and the presence or absence of recently suggested minor ECG criteria (SV22.5 mV, pathological Q waves, small Q-waves (>0.1 mV in two adjacent leads), and complete or incomplete bundle branch blocks (BBB) were registered [16,17]. The two-dimensional echocardiographic recordings were analysed with respect to: left ventricular end-diastolic and end-systolic diameter (LVEDD, LVESD), left atrial diameter (LA), maximal diastolic septal thickness (MaxIVS), left ventricular posterior wall diastolic thickness (LVPWT); and Doppler recordings of: left ventricle isovolumetric relaxation time (LVIRT), as well as E and A wave flow velocity ratio (E/A ratio). In each family, based on the presence or absence of one or more additional family members fulfilling diagnostic criteria the occurrence of FHC was classified as familial or indeterminable inheritance. In each family, presence of major cardiac events were registered, i.e. sudden cardiac death before the age of 40 years (including patients resuscitated from cardiac arrest); presence of symptomatic children with FHC (≤15 years of age); severely symptomatic adults with NYHA-functional classification scores ≥3, need for DDD pacemaker implantation, myectomy or percutaneous transluminal septal myocardial ablation (PTSMA). One hundred randomly selected anonymous Guthrie cards [20] from persons born in Denmark were used as genetic controls (Local Science Ethics Committee Copenhagen, protocol no. 01-119/99).

2.2 Mutation detection
Genomic DNA was extracted from blood samples using a QIAamp DNA purification kit (Qiagen, Germany). Mutation analysis of the MYH7 gene was limited to exons 3–23 as all but one of the previously described mutations are located in these exons [21]. Genomic DNA was PCR-amplified using intronic primers and subjected to single strand conformation polymorphism–heteroduplex (SSCP–HD) analysis as described [20]. Aberrant conformers were sequenced on an ABI373 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Recent studies have shown that more than one mutation may be present in a FHC family [22,23]. On this basis, all probands with a MYH7 gene mutation identified were screened for mutations by a combination of SSCP–HD and direct sequencing in other FHC associated genes (MYBPC3, MYL2, MYL3, TPM1, TNNT2, TNNI3 and ACTC) as described [4–8].

2.3 Statistics
Continuous data were analysed using ANOVA (Statistic Package STATISTICA for Windows, StatSoft, Inc. (1999) version 5.5; Tulsa, OK, USA). Normal plots were used to asses normality of distributions. Fisher's exact test and the Mann–Whitney test were used to analyse non-continuous data. A P value<0.05 was considered significant and all tests were two-sided. Data are presented as means with standard deviations (mean (S.D.)), unless otherwise indicated.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Genotype-phenotype...
 5. Discussion
 6. Conclusion
 References
 
Demographic and clinical data of the 68 probands are presented in Table 1. There was a small overrepresentation of males (59%) as well as of familial occurrence (see Section 2) of FHC (59%). The large majority (90%) of probands had the predominantly proximal septal type of hypertrophy. There was a general tendency to left atrial enlargement, with preserved left ventricular cavity dimensions and borderline hypertrophy of the left ventricular posterior wall.


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  		Table 1 Characteristics of probands with hypertrophic cardiomyopathy (n=68)

 
3.1 MYH7 mutations
We identified eight different MYH7 gene mutations in nine unrelated families. Four of these mutations were identified for the first time in this study, i.e. Leu601Val, Val320Met, Asp778Glu and Glu846Gln and four of the mutations have previously been reported with pedigrees and clinical data, i.e. Arg190Thr [20], Arg694Cys [24], Leu390Val [25], and Val606Met [26]. The diagnosis of FHC and mutation segregation in pedigrees was based on the major diagnostic criteria [15–17]. The mutations (nucleotide changes) and the corresponding amino acid substitutions in the MYH7 gene are listed in Table 2. The mutations identified were located in different exons throughout the exons analysed (3–23) and all were missense mutations.


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  		Table 2 Cardiac β-myosin heavy chain gene (MYH7) mutations identified in Danish hypertrophic cardiomyopathy patients

 
3.2 Clinical presentation of the mutations identified
Pedigrees of families carrying the mutations Leu601Val (ZO), Val320Met (XD), Asp778Glu (XI) and Glu846Gln (ZA) are shown in Fig. 1 and echocardiographic and ECG data of individual mutation carriers are presented in Table 3.


Figure 1
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  		Fig. 1 Pedigrees of families with FHC associated with MYH7 gene mutations. Filled symbols: clinically and genetically (MYH7 gene mutation) affected; black dot: mutation (MYH7) carriers; ?: not available for inclusion/not examined; arrow: probands; *: carriers of MYBPC3 gene mutation (see text).

 

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  		Table 3 Echocardiographic and ECG indices in MYH7 gene mutation carriers

 
3.2.1 Leu601Val mutation
The Leu601Val mutation was identified in proband ZO:III-1. She was diagnosed at the age of 7 years when suffering from syncopes. At the age of 15, she was myectomized (modified Konno procedure) after sudden deterioration of cardiac function associated with a high left ventricular outflow tract pressure gradient (95 mmHg), following an acute infection. At the age of 18, she developed a 2° AV-block with recurrence of syncopes, and a DDD-pacemaker was implanted. Neither the proband's parents, their siblings nor the proband's sister had any signs of myocardial hypertrophy or cardiac symptoms, and none of them carried the mutation. Thus, the Leu601Cys had arisen de novo in the proband, as paternity was genetically confirmed.

3.2.2 Val320Met mutation
The Val320Met mutation was identified in proband XD:II-1 at the age of 42 years. She suffered from dyspnoea and syncope on exertion, and had a high (>100 mmHg) left ventricular outflow-tract gradient at rest with a further increase during exercise. At the age of 44 years, she was successfully treated with PTSMA. None of the proband's relatives fulfilled diagnostic criteria for hypertrophic cardiomyopathy or had any cardiac symptoms, but the presence of a proximal ‘septal bulb’ configuration in her asymptomatic 15 year old daughter (XD:III-2) was suggestive of her being a mutation carrier, and she was subsequently identified as such, whereas the proband's 42 year old brother (XD:II-2) also carrying the mutation had neither cardiac signs nor symptoms.

3.2.3 Asp778Glu mutation
The Asp778Glu mutation was identified in proband XI:IV-3. She was diagnosed during her first pregnancy at the age of 36 years, when she developed dyspnoea and angina. β-Blocker therapy effectively relieved symptoms. The family history revealed two cases of sudden deaths at the age of 39 years (XI:II-3) and 40 years (XI:III-3), respectively. XI:III-3 was obligate mutation carrier. XI:III-1 had septal hypertrophy, left atrial dilatation and atrial fibrillation. XI:IV-1 and XI:IV-4 both fulfilled diagnostic criteria for FHC with asymmetric septal hypertrophy but were without symptoms. XD:IV-2 had septal hypertrophy, a mid-ventricular outflow gradient, episodes of dizzy spells, and episodes of non-sustained ventricular tachycardia on 24-h Holter monitoring. On this basis—including the family history of sudden death—an ICD unit was implanted. The screening for mutations in other FHC-associated genes in this family identified one additional mutation/polymorphism in the MYBPC3 gene (Val896Met) previously associated with FHC [27]. Carriers of this additional mutation/polymorphism are indicated in the pedigree. Thus, XI:III-1, III-3 (obligate carrier), IV-1, IV-3 and IV-4 were double heterozygous for the two mutations. Family members with both mutations as well as those with only the MYH7 mutation were all clinically affected.

3.2.4 Glu846Gln mutation
The Glu846Gln mutation was identified in proband ZA:II-8. She had suffered from angina and dyspnoea since the age of 58 years. After medical treatment and DDD-pacemaker implantation had failed to relieve a high LVOT-gradient, she was successfully myectomized. Two sisters (ZA:II-6 and ZA:II-12) also fulfilled diagnostic criteria for FHC, but both were without symptoms. Other family members had neither signs of myocardial hypertrophy nor cardiac symptoms. Thus, ZA:III-5 (aged 42 years); III-7 (aged 39 years); III-12 (aged 32 years) and IV-6 (aged 19 years) all carrying the mutation were non-penetrant.

3.3 The combined presence of MYH7 gene mutations and MYBPC3 gene mutations/polymorphisms
In addition to the MYBPC3 gene mutation/polymorphism identified in family XI (MYBPC3, Val896Met) the mutation screening of other sarcomere genes identified two different MYBPC3 gene mutations/polymorphisms in two of the families with MYH7 gene mutations previously reported [24], i.e. a probable splice site mutation (g10899 IVS16 -6 G>A) in family G (MYH7, Arg694Cys) and Ala833Thr (g16153G>A) in family E (MYH7, Leu390Val) (data not shown) [25]. None of these mutations have been identified before and they were not present in any of the 100 genetic controls.


   4. Genotype–phenotype relationship
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Genotype-phenotype...
5. Discussion
 6. Conclusion
 References
 
4.1 MYH7 gene mutation probands vs. non-MYH7 gene mutation probands
Between adult probands with a MYH7 gene mutation (MYH7 probands (n=9)) and probands without a MYH7 gene mutation (non-MYH7 probands (n=59)) no significant differences were present with respect to sex distribution, height or blood pressure. However, a higher proportion of women among the MYH7 probands (67 vs. 37%; ns) may have resulted in the tendency to a lower body weight (66 (15) vs. 76 (15) kg; ns) in the MYH7 group. None of the MYH7 probands had hypertrophy of the apical or predominantly mid-ventricular types. Myectomy or PTSMA had been performed in a higher proportion of the MYH7 probands (56 (5/9) vs. 17% (10/59); P<0.05), and MYH7 families overall had a higher rate of major cardiac events (see Section 2) as compared to the non-MYH7 families (89 (8/9) vs. 42% (25/59); P<0.05). The distribution of familial and indeterminable inheritance in the MYH7 and non-MYH7 families was not significantly different. Except for a significantly lower MaxIVS thickness in MYH7 probands (18 (4) vs. 22 (4) mm; P=0.03), no other 2D-echocardiographic parameters differed significantly between the two groups. The trans-mitral E/A flow-ratio was significantly higher in the MYH7 probands (1.9 (0.5) vs. 1.3 (0.7); P=0.049) whereas no difference was observed in LVIRT. No significant differences were identified when comparing the ECG scores from MYH7 and non-MYH7 probands. These results were not altered when the three MYH7 probands with concomitant MYBPC3 gene mutations/polymorphisms were excluded from the analyses. Thus, within the group of probands, ECG and echocardiography did not distinguish between non-MYH7 and the MYH7 probands who had experienced high rates of cardiac events in their families.

4.2 Families with a MYH7 gene mutation: mutation carriers vs. non-carriers
In order to evaluate the outcome of screening based on clinical diagnostic criteria in relatives to MYH7 probands, we compared adult mutation-carriers (above 15 years of age) to their adult non-carrier family members. There were no significant differences between the two groups with respect to sex distribution, age, height, weight or blood pressure. Between carriers and non carriers there were no significant differences with respect to left ventricular cavity dimensions, whereas values for LA (42 (11) vs. 35 (4) mm; P=0.001), MaxIVS (16 (5) vs. 10 (3) mm; P<0.001) and LVPWT (10 (2) vs. 9 (2) mm; P=0.047) were significantly increased in carriers. There was a longer LVIRT in the mutation carriers (0.07 (0.02) vs. 0.06 (0.02) s; P=0.02), whereas the E/A ratio did not differ between the two groups. However, further analysis disclosed substantial overlap of values of these overall significantly different parameters between the two groups (Fig. 2).


Figure 2
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  		Fig. 2 Echocardiographic findings in hypertrophic cardiomyopathy families associated with MYH7 gene mutations, divided between mutation carriers (yes) and family members without mutation (no). Individual values are indicated. The mean values (bold lines) of the four parameters shown are all significantly different between the two groups. Thin lines indicate proposed [15,17] diagnostic cut-of values. Abbreviations: LA, left atrium diameter; MaxIVS, maximal inter ventricular septal thickness (diastole); LVPWT, left ventricular posterior wall thickness (diastole); LVIRT, left ventricular isovolumetric relaxation time.

 
The ECG findings in mutation carriers and non-carriers are listed in Table 4. A Romhild–Estes score above 3, fulfillment of Sokolow criteria and the presence of small Q waves were found significantly more frequent in the MYH7 mutation carriers than non-carriers. Except for a Romhild–Estes score above 3 the positive predictive values (PPV) were generally low. In addition, negative predictive values (NPV) were also low reflecting the overall low diagnostic sensitivity in addition to the low specificity of these ECG criteria. No non-carriers had pathological Q-waves in their ECG; however, only three carriers (8%) had pathological Q-waves. In the family screening, a MaxIVS ≤15 mm did not distinguish between mutation carriers and non-carriers (Fig. 2). In order to assess the diagnostic gain from the ECG parameters in these cases, we further analysed the proportions of genotyped family members with a MaxIVS ≤15 mm fulfilling the different diagnostic ECG criteria in this subset of adult MYH7 family members (Table 4). Only the presence of small Q waves was significantly different in this sub-analysis. Again, none of the non-carriers had a Romhild–Estes score of 3 or more or pathological Q waves, but only one (8%) and two (15%) of the mutation carriers fulfilled these criteria, respectively. Thus, the presently assessed ECG criteria do not seem to add to the refinement of the diagnosis of FHC in the subset with little or no hypertrophy on echocardiography.


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  		Table 4 Major and minor ECG findings in families with a MYH7 gene mutation

 
4.3 Cardiac events and outcome of clinical and genetic screening in families with a MYH7 gene mutation
The cardiac events in the MYH7 families are summarized in Table 5. Ten confirmed and six probable mutation carriers had experienced at least one major cardiac event. There were major cardiac events in all but one of the nine families. The penetrance of the MYH7 gene mutations was reduced, as 11 of 38 mutation-carriers did not fulfill echocardiographic diagnostic criteria at the time of inclusion in the study. However, this reduced penetrance is partially explained by the observation that four of the non-penetrant mutation-carriers were children and only two were older than 40 years of age (mean 26; range 8–52 years). In addition, in the group of non-carriers, six family members with hypertrophy on echocardiography, but also demonstrating additional possible etiological explanations for the hypertrophy could be correctly classified and excluded as mutation-carriers, i.e. five patients treated for hypertension for more than one year, and two obese patients with a body mass index <33. Thus, the genetic analyses allowed a clear distinction between the 27 mutation-carriers with hypertrophy caused by FHC and the six non-carriers with secondary hypertrophy. Moreover, in the group of 63 relatives with a normal echocardiographic phenotype (including 23 children), but at risk for carrying a mutation, only 11 were found to be carriers. Thus, out of the 63 echocardiographically normal relatives the 52 non-carriers (83%) could be excluded from further follow up after the genetic diagnosis was established.


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  		Table 5 Number of major cardiac events, symptomatic mutation-carriers and the fulfillment of diagnostic echocardiographic criteria in nine families with FHC caused by MYH7 gene mutations

 

   5. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Genotype-phenotype...
 5. Discussion
6. Conclusion
 References
 
For the first time, the outcome of genetic screening as an extension of the recommended clinical screening of relatives to probands with a MYH7 gene mutation was assessed in a consecutively included cohort. In the nine MYH7 families, the presence of two cases of sudden death at the age of 14 years and a total of five cases of sudden death before the age of 30 years indicates the need for family screening. The study demonstrated that in the group of relatives at risk for carrying a mutation, but without clinical signs of FHC 83% could unambiguously be identified as healthy. This negative test result is of outmost importance for these relatives for several reasons; the clinical screening follow-up can immediately be terminated and the psychological, occupational and legal restrains that may be associated with the risk for being a carrier are definitely removed. Furthermore, the genetic screening could correctly identify the subset of relatives with myocardial hypertrophy due to a different aetiology. Finally, the genetic screening identified a small group of relatives at high risk for development of FHC and follow-up of these relatives can be intensified accordingly.

5.1 MYH7 gene mutations
The previously reported frequency of MYH7 gene mutations in FHC has ranged from 30 to 50% in the initial studies [28,29] to 10–20% most recently [2,3,30]. In our FHC population, including both tertiary referral centre patients and an additional local area population, the frequency of FHC families with a MYH7 gene mutation was found to be 13%. This number may represent a small underestimate of the true number of FHC patients/families with MYH7 gene mutations, since the mutation detection techniques rely on mutation screening with the SSCP/HD analysis. We have previously reported a detection rate up to 100% with these screening methods [31]. However, by these methods, large deletions affecting full exons or more will be missed, and we have not examined the exons 24–40 encoding the β-myosin tail. However, the few comparable studies have used a similar approach [2,3]. Our presently and previously identified MYH7 gene mutations are all believed to be pathogenic. This is substantiated by the observation that they all occur in highly conserved regions/residues in cardiac and skeletal muscle myosins (for myosin alignment database see: http://www.mrc-lmb.cam.ac.uk/myosin/myosin.html), segregated with the disease in the family [20,25,26] or had arisen de novo in the proband and were not identified in the 100 controls or any of the other 66/67 FHC probands, i.e. in a total of more than 300 chromosomes. In addition, a mutation in codon 778, Asp778Gly, has previously been associated with FHC with a high penetrance in several Japanese FHC families [32].

The presence of the MYBPC3 gene mutations or rare polymorphisms (MYBPC3; Val896Met, Ala833Thr and g10899 IVS16 -6 G>A) in three families may have affected the phenotypic presentation. However, as no carriers of these MYBPC3 gene mutations fulfilled diagnostic criteria for FHC without the simultaneous presence of the MYH7 gene mutation, the two mutations were not independently associated with disease in these families. In addition, there was no obvious relationship between the degree of hypertrophy and the presence of one or two of the mutations. The finding of these three MYBPC3 gene mutations in three of nine MYH7 families emphasizes the need for inclusion of all sarcomere genes in mutation screening. In addition, when identifying a novel mutation in a small family, where the assessment of co-segregation with the disease is difficult, the search for other mutations should be continued. The small size of some of the MYH7 families causes difficulties in the assessment of disease and mutation segregation. The inclusion of FHC probands with a relatively small number of family members available is a consequence of the consecutive design of the study. However, this reflects a clinical setting that is often faced. That two of the MYH7 gene mutations were identified in probands without clinical evidence of familial disease, but later proven to be familial by the identification of the mutation, also shows that additional clinical genetic counselling should rely on molecular genetic diagnostics whenever they can be obtained. At present, screening for mutations are still a very laborious task. However, the use of high throughput mutation detection techniques, e.g. automated PCR robots, automated capillary electrophoresis SSCP and eventually chip-based technology—may in the near future make genetic analyses much more readily available [31,33].

5.2 Genotype–phenotype: MYH7 mutation vs. non-MYH7 mutation probands
None of the presently evaluated echocardiographic and ECG parameters seem to allow a distinction between MYH7 and non-MYH7 mutation probands when deciding which genes to examine. However, as the total number of MYH7 probands was relatively small, studies with larger numbers of genotyped (MYH7) families are needed to clarify whether these observations together with the significantly higher rate of major cardiac events in the MYH7 families (see below) and the absence of MYH7 gene mutations in probands with apical hypertrophy applies in general. The identification of XD:IV2 with the predominantly mid-ventricular phenotype associated with a mutation located in the region of myosin and myosin light chain interaction (Asp778Glu) is interesting. Mutations in the light chain genes have previously been associated—although not exclusively—with this specific and rare form of FHC [3,6].

5.3 Clinical screening vs. genetic analyses
The large overlap of values of all four significantly different echocardiographic parameters (Fig. 2), as well as the low sensitivity and specificity of various ECG parameters in family members with no or little hypertrophy, generally demonstrate the inability of conventional diagnostic tools to exclude the diagnosis of FHC in family screening programs.

With respect to ECG, only the Romhild–Estes score and the presence of Q waves had 100% specificity in the families examined. However, in the subset of MYH7 family members with a MaxIVS ≤15 mm where the overlap in echocardiographic parameters is present, the importance of this finding is low, as the diagnostic sensitivities were only 7 and 14%, respectively. All other ECG parameters resulted in false positives if used to screen family members, and only the difference in the presence of small Q waves was significant. However, the diagnostic value of this finding is impaired by the low NPV and a PPV of only 56% indicating that only little more than half of the persons in this echocardiographic ‘gray-zone’, in whom small Q waves are found, will be correctly classified. Recently, echocardiographic Doppler tissue imaging of mutation carriers without hypertrophy in a rabbit model of FHC has shown excellent sensitivity and specificity for identifying mutation carriers [34]. An early human study has found a comparable diagnostic benefit from the use of echocardiographic Doppler tissue imaging [35], whereas a recent study has been less convincing [36]. As shown in Table 5, there were major cardiac events in all but one small MYH7 family, including seven sudden deaths at young age. The majority of the family members were aware of the serious events often associated with FHC. The genetic assessment reduced the group of possible non-penetrant mutation carriers from 63 to 11, i.e. by 83%. Therefore, the majority of these family members generally perceived the genetic diagnosis as very reassuring; and in addition, a more appropriate follow-up of the remaining 17% could be planned for these (young) non-penetrants who may develop hypertrophy and symptoms later in life. However, pre-symptomatic genetic screening may in patients found to carry a mutation cause psychological adverse effects. Additional adverse outcomes of genetic screening may include social, occupational and insurance matters in persons who may never develop the disease. Formal assessment of these aspects is not available. Generally, expert clinical genetic counselling should precede pre-symptomatic genetic testing. For other inherited cardiac diseases, cardiologists have recommended different follow-up regimes depending on whether the diagnosis was based on genetic findings—if available—or when the diagnosis was based on clinical findings alone [37]. No consensus for follow-up recommendations in FHC based on genetic findings is available at present. However, reports of large genotyped cohorts may improve the prognostic use of genetic information. In addition, efforts—including randomised studies—to change the natural history of FHC disease progression, e.g. long term medical therapy of young and possibly non/‘low’-penetrant family members, would have to rely on firm diagnostic—genetic—findings.


   6. Conclusion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Genotype-phenotype...
 5. Discussion
 6. Conclusion
References
 
A mutation in the MYH7 gene was found in 13% of the FHC families. MYH7 families had a high rate of major cardiac events, but no other specific MYH7 phenotypic characteristic were identified. In relatives with little or no hypertrophy on echocardiography, minor clinical diagnostic criteria alone were not useful for identification of mutation carriers. The inclusion of genetic analyses offered a marked diagnostic advantage as compared to clinical screening alone as the genetic diagnosis reduced the number of possible non-penetrant mutation carriers needing follow-up by 83% and 17 relatives (18%) in the MYH7 families examined were either false positive or false negative for the FHC diagnosis as assessed by echocardiography. Studies of psychological, social and occupational and other possible adverse outcomes of pre-symptomatic genetic testing are needed.

Time for primary review 28 days.


   Acknowledgements
 
This work was supported by grants from The Danish Heart Foundation, Director Emil C. Hertz and wife Inger Hertz's foundation, Danish Medical Association Research Foundation, H:S–Copenhagen Hospital Corporation and The Danish Medical Research Council. We are grateful to Anders Kirstein Pedersen, MD and Anders Dupont Børglum, MD for valuable discussions, expertise and support.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Genotype-phenotype...
 5. Discussion
 6. Conclusion
 References
 

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