Diagnostic value of abnormal Q waves for identification of preclinical carriers of hypertrophic cardiomyopathy based on a molecular genetic diagnosis

Tetsuo Konno*, Masami Shimizu, Hidekazu Ino, Masato Yamaguchi, Hidenobu Terai, Katsuharu Uchiyama, Kotaro Oe, Tomohito Mabuchi, Tomoya Kaneda and Hiroshi Mabuchi

Molecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science, Kanazawa University, Kanazawa Japan

* Correspondence to: Tetsuo Konno, MD, Molecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science, Kanazawa University, Takara-machi 13-1, Kanazawa 920-8640, Japan. Tel: +81-76-265-2254, Fax: +81-76-234-4251
E-mail address: inmed-i{at}p1.tcnet.ne.jp

Received 13 May 2003; revised 20 October 2003; accepted 30 October 2003

Abstract

Aims There are currently no established diagnostic criteria for the identification of abnormal Q waves in patients with hypertrophic cardiomyopathy (HCM), resulting in various definitions being applied in each previous study. The aim of this study was to determine the most accurate diagnostic definition of abnormal Q waves for HCM based on a molecular genetic diagnosis, and also to apply abnormal Q waves to the identification of preclinical carriers.

Methods and results We applied three different criteria used in previous reports for abnormal Q waves in 148 genotyped subjects. Of the three criteria, Criterion 3 (Q wave >3mm in depth and/or >0.04s in duration in at least two leads except aVR) showed the highest sensitivity (50% in the young, 29% in adults) while retaining a high specificity (90% in the young, 97% in adults), resulting in the highest accuracy (69% in the young, 52% in adults). Using Criterion 3, abnormal Q waves were present 27.6% of preclinical carriers, and in 5.4% of non-carriers (P<0.01).

Conclusions These findings suggest that Criterion 3 may be the most accurate diagnostic definition for HCM. Understanding the diagnostic value of abnormal Q waves may be useful in screening preclinical carriers of HCM.

Key Words: Hypertrophic cardiomyopathy • electrocardiography • genetics

1. Introduction

Hypertrophic cardiomyopathy (HCM) is a primary cardiac disorder, often transmitted genetically, with a heterogeneous clinical and morphological expression.1The diagnosis of HCM is conventionally based on the echocardiographic demonstration of unexplained left ventricular hypertrophy (LVH).2In clinical practice, the identification of preclinical carriers is of significant importance because sudden death occurs even in young, asymptomatic patients with HCM.3However, the clinical diagnosis of HCM is particularly difficult in clinically healthy subjects, especially in the young, because ventricular wall hypertrophy may not be apparent until adulthood. Recent molecular genetics studies in HCM have provided a strategy to identify healthy carriers of disease-causing mutations. Although genetic analysis has become widely available, it is still difficult to make the genetic diagnosis in all patients with HCM because of the genetic heterogeneity of the disease. Therefore, clinically accessible methods to distinguish healthy carriers from non-carriers are required even in the post-molecular era. From this point of view, electrocardiography (ECG) is a clinically accessible, simple and cost-effective method forscreening clinically healthy subjects for HCM.4Previous studies have demonstrated that ECG abnormalities may be observed preceding the appearance of ventricular wall hypertrophy in carriers with disease-causing mutations for HCM.4Abnormal Q waves are one of the most common ECG abnormalities in HCM, occurring in 20 to 50% of patients, and occur most frequently in young patients with HCM.5Interestingly, it is reported that abnormal Q waves may be found first and at a higher frequency compared with other ECG abnormalities such as LVH and ST-T abnormalities in carriers with a certain disease-causing mutation.6These findings suggest that the appearance of abnormal Q waves may predict the genetic status in preclinical carriers with HCM. However, there are currently no established criteria for the identification of abnormal Q waves in HCM, resulting in various definitions of abnormal Q waves being applied in each previous study. Furthermore, no report has investigated the diagnostic value of abnormal Q waves in preclinical patients with HCM based on a molecular genetic diagnosis. The purpose of the present study was to determine the most accurate diagnostic definition of abnormal Q waves for HCM based on a molecular genetic diagnosis, and also to apply abnormal Q waves to the identification of preclinical carriers in clinically healthy subjects.

2. Methods

2.1. Subjects
Fifteen families with HCM in which the disease-causing mutation was identified were studied. After the mutation was identified in the proband with HCM, the family members were studied by a 12-lead ECG and echocardiography and blood samples were obtained for genetic analysis. A total of 150 subjects were included in this study. One subject was excluded because of a history of an old anterior myocardial infarction, and a second subject was excluded because of pre-excitation, leaving 148 for analysis. Informed consent was obtained from all subjects or their guardians in accordance with the guidelines of the Bioethical Committee on Medical Researches, School ofMedicine, Kanazawa University.

2.2. Detection of mutations
DNA of the probands was isolated from peripheral white blood cells, as previously described.7Amplification of genomic DNA was performed using the polymerase chain reaction. Oligonucleotide primers were used to amplify exons of the cardiac myosin binding protein-C gene, beta-myosin heavy chain gene, cardiac troponin T gene and cardiac troponin I gene as previously reported.8–11Single-strand conformational polymorphism (SSCP) analysis of amplified DNA was then performed. For abnormal SSCP patterns, the nucleotide sequences of the cloned PCR products were determined on both strands by the dye terminator cycle sequencing method with use of an automated fluorescent sequencer (ABI PRISMTM 310 Genetic Analyzer, PE Biosystems, Foster City, Calif). The sequence variation was confirmed by restriction enzyme digestion. The same method was then used to determine the genotype in DNA from family members of the probands

2.3. Criteria of abnormal Q waves
A standard 12-lead ECG was recorded in all subjects in the supine position during quiet respiration. The criteria for abnormal Q waves were defined as follows based on previous studies: (Criterion 1) Q wave >1/3 of the ensuing R wave in depth and/or >0.04s in duration in at least two leads except aVR,12(Criterion 2) Q wave >1/4 of the ensuing R wave in depth and/or >0.04s in duration in at least two leads except aVR,4(Criterion 3) Q wave >3mm in depth and/or >0.04s in duration in at least two leads except aVR.13Because the Q wave duration (> 0.04s) is the same for all three criteria, we actually interpreted the Q wave findings as follows: (1) When no wide Q waves and narrow Q waves in >2 leads were observed, we evaluated the findings as to whether the depth of the narrow Q waves fit each criterion. (2) When a wide Q wave (of any depth) in only one lead and narrow Q waves in other leads were observed, we evaluated the findings as to whether the depth of the narrow Q waves in the other leads fit each criterion. (3) When wide Q waves (of any depth) were observed in >2 leads, it was diagnosed as the existence of abnormal Q waves in all criteria. ECG abnormalities other than abnormal Q waves were defined as follows: LVH assessed by a Romhilt–Estes score >=4,14ST-segment depression of an upsloping type >0.1mV at 0.08s after the J point, or those of horizontal or downsloping type >0.05mV,6T-wave inversion >0.1mV except aVR and V1 to V2 leads in the absence of conductiondisturbance.6

2.4. Echocardiographic criteria
Standard M-mode and two-dimensional echocardiographic studies were performed to identify and quantify morphologic features of the left ventricle. Left ventricular dimensions and the thickness of the septum and posterior wall were measured at the level of the tips of the mitral valve leaflets. The fractional shortening was calculated as the difference in end-diastolic and end-systolic dimensions. Left ventricular maximum wall thickness 13mm in adults or 95% CI of the theoretic value in children were considered the diagnostic criteria for HCM.15

2.5. Statistical analysis
Sensitivity was defined in percent as true-positives/(true-positives+false-negatives)x100; specificity as true-negatives/(true-negatives+false-positives)x100; positive predictive value as true-positives/(true-positives+false-positives)x100; negative predictive value as true-negatives/(true-negatives+false-negatives)x100; accuracy as (true-positives+true-negatives)/(true-positives+true-negatives+false-positives+false-negatives)x100.16Continuous data were expressed as means±SD and were analysed with unpaired, two-tailed t-tests. Categorical data were compared with chi-square tests. Sensitivity was compared with McNemar's chi-square test. Differences were considered to be statistically significant at P value <0.05.

3. Results

3.1. Genetic results and characteristics of subjects
Nine different mutations were identified in 15 families. Genetic analysis revealed that 92 of the 148 subjects enrolled were genetically affected, and 56 subjects were genetically unaffected. Of the 92 genetically affected subjects, 18 were associated with the cardiac myosin binding-protein C gene mutation (Arg820Gln, n=14; Del593C, n=4); six were associated with the beta-myosin heavy chain gene mutation (Ala26Val, n=3; Glu935Lys, n=3); 21 were associated with the cardiac troponin T gene mutation (Arg92Trp, n=8; Lys273Glu, n=10; Val85Leu, n=1; Phe110Ile, n=2); and 47 were associated with the cardiac troponin I gene mutation (Lys183del; n=47). All mutations have been previously identified and described elsewhere.7,17–21The mean age of the 92 genetically affected subjects was 45±21 years (range; 2 to 88 years), and the male/female ratio was 43/49 (46.7% and 53.3%, respectively). The number of genetically affected subjects aged <30 years was 24 and >=30 years was 68. The mean age of the 56 genetically unaffected subjects was 42±22 years (range; 6 to 86 years), and the male/female ratio was 28/28 (50% and 50%, respectively). The number of the genetically unaffected subjects aged<30 years was 21 and>30 years was 35. Six subjects (three genetically affected and three genetically unaffected) had a history of hypertension.

3.2. Diagnostic value of various criteria of abnormal Q waves
Table 1shows the diagnostic value of the various criteria for abnormal Q waves for carriers with disease-causing mutations. In both the young and adult populations, Criterion 3 showed the highest sensitivity (50% in the young, 29% in adults) while retaining a high specificity (90% in the young, 97% in adults), resulting in the highest accuracy (69% in the young, 52% in adults). In the adult population, the sensitivity of Criterion 3 was significantly higher than that of Criterion 1 (29% vs 19%, P<0.05). All three criteria showed higher diagnostic values in the young population in comparison with that in the adult population, with a higher sensitivity and a higher accuracy, but not much change in specificity. In all subjects, the sensitivity of Criterion 2 and Criterion 3 was significantly higher than that of Criterion 1 (Criterion 2 vs Criterion 1; 32% vs 23%, P<0.05, Criterion 3 vs Criterion 1; 35% vs 23%, P<0.01). Overall, in all subjects (n=148), the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of Criterion 3 were 35%, 95%, 91%, 47% and 57%, respectively. When the six hypertensive subjects (three genetically affected and three genetically unaffected) were excluded, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of Criterion 3 in the remaining subjects (n=142) were 34%, 94%, 91%, 47% and 57%, respectively. The diagnostic value of Criterion 3 was not altered by exclusion of the hypertensivesubjects.


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Table 1 Diagnostic value of various criteria of abnormal Q waves for carriers with disease-causing mutations

 
3.3. Diagnostic value of ECG abnormalities
Table 2shows the diagnostic value of the major ECG abnormalities for carriers with disease-causing mutations. The definition of abnormal Q waves was made using Criterion 3, which showed the most accurate diagnostic value for HCM (Table 1). In the young population, abnormal Q waves showed the highest sensitivity (50%), with a high specificity (90%) among the major ECG abnormalities. On the other hand, in the adult population, abnormal Q waves showed the lowest sensitivity (29%), a high specificity (97%) and the lowest accuracy (52%). In all subjects, abnormal Q waves showed the lowest sensitivity and the lowest accuracy, while the specificity was similar to the other major ECG abnormalities. When the major ECG abnormalities were combined (abnormal Q and/or ST-T and/or LVH), the sensitivity, negative predictive value and accuracy were improved while retaining a high specificity and positive predictive value in both the young and adult populations. Overall, in all subjects (n=148), the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of combination of major ECG abnormalities were 84%, 86%, 91%, 76% and 84%, respectively.


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Table 2 Diagnostic value of ECG abnormalities for carriers with disease-causing mutations according to agea

 
3.4. Age-related frequencies of abnormal Q waves
The frequency of abnormal Q waves (by Criterion 3) in different decades of life are shown in Fig. 1. In carrier subjects, abnormal Q waves were most frequently found in teens, and gradually decreased after 20 years of age, whereas the frequency of left ventricular wall hypertrophy demonstrated by echocardiography increased after 20 years of age. The youngest carrier having abnormal Q waves was a 7-year-old boy with a deletion of Lysine 183 mutation in the cardiac troponin I gene.



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Fig. 1 Age-related frequencies of abnormal Q waves (by Criterion 3) and echocardiographic abnormalities. Solid bars, percentage of carriers with abnormal Q waves; open bars, percentage of carriers with left ventricular wall hypertrophy demonstrated by echocardiography.

 
3.5. Abnormal Q waves in preclinical carriers
Abnormal Q waves were applied to the identification of preclinical carriers in clinically healthy subjects without LVH on echocardiography. Of the 92 genetically affected subjects, 29 did not manifest LVH on echocardiography, and they were defined as preclinical carriers. Of the 56 genetically unaffected subjects, one hypertensive subject was excluded because of the presence of LVH on echocardiography. The frequency of abnormal Q waves (Criterion 3) was significantly higher in preclinical carriers than in non-carriers (27.6% in preclinical carriers; n=29, 5.4% in non-carriers; n=55, P<0.01). The maximal wall thickness (MWT) and age were not significantly different between preclinical carriers and non-carriers (MWT; 9.4±2.0mm in preclinical carriers, 9.5±1.4mm in non-carriers, P=ns, age; 31.1±22.8 years in preclinical carriers, 41±21.7 years in non-carriers, P=ns). In screening for preclinical carriers in clinically healthy subjects without LVH, the sensitivity, specificity, positive predictive value, negative predictive value and accuracy of abnormal Q waves (by Criterion 3) were 28%, 95%, 73%, 71% and 71%, respectively.

4. Discussion

This study demonstrated that Criterion 3 for abnormal Q waves was the most accurate for diagnosing carriers associated with HCM, and that abnormal Q waves might be one of the clinically accessible methods to identify preclinical carriers in clinically healthy subjects.

4.1. Diagnostic value of abnormal Q waves in HCM
In young subjects, we demonstrated that Criterion 3 (Q wave >3mm in depth and/or >0.04s in duration in at least two leads except aVR) showed the highest sensitivity with a favourable specificity, resulting in the highest accuracy. This criterion has been used as a diagnostic criterion for HCM in children.13On the other hand, in adult populations, previous studies4–12have defined abnormal Q waves for HCM as >1/4 or 1/3 of the ensuing R waves in depth and/or >0.04s in duration (in our study, Criterion 1 or 2, respectively). Interestingly, the diagnostic value of Criterion 3 was the most accurate of the three, not only in the young population but also in the adult population, with the highest sensitivity without loss of specificity. Using Criterion 3 in the adult population, we were able to detect seven additional adult carriers compared with results using Criterion 1, and twoadditional adult carriers compared with results using Criterion 2, whereas the number of false positives did not increase (one false positive in Criterion 2, no false-positive in Criterion 1 and 3). From these findings, we propose that Criterion 3 may be the most accurate diagnostic definition in screening not only the young population, but also the adult population for carriers with disease-causing mutations.

In all subjects, in comparison with other major ECG abnormalities abnormal Q waves showed the lowest sensitivity and accuracy, while the specificity was similar to LVH and ST-T abnormality. Similar results were observed in the adult population. However, in the young population, abnormal Q waves showed the highest sensitivity and a high specificity. These findings suggest that the diagnostic value of major ECG abnormalities may differ according to the age of the population being studied. Thus, in screening for carriers with HCM on ECG, it is important to consider the age of the subjects in interpreting major ECG abnormalities.

4.2. Diagnostic value of abnormal Q waves in preclinical carriers
In genotyped families, we can easily identify preclinical carriers using molecular genetic methods. On the other hand, in ungenotyped families, the genetic heterogeneity of HCM makes it difficult to identify preclinical carriers.22Therefore, it is still of prime importance to investigate the ECG findings of preclinical carriers, even in the post-molecular era.

In this study, we observed abnormal Q waves even in carriers without left ventricular wall hypertrophy demonstrated by echocardiography. From these findings, the hypothesis is advanced that appearance of abnormal Q waves may predict the genetic status in preclinical patients with HCM. To test this hypothesis, we applied an evaluation of abnormal Q waves to the identification of preclinical carriers in clinically healthy subjects. No study has performed this type of investigation. Interestingly, the frequency of abnormal Q waves was more than five-times higher in preclinical carriers than in non-carriers (27.6% in preclinical carriers, 5.4% in non-carriers, P<0.01). These findings suggest that abnormal Q waves may be one of the useful methods to identify preclinical carriers in clinically healthy subjects. Although the pathogenesis of abnormal Q waves in preclinical carriers has not been fully clarified, Shimizu et al. postulated a mechanism whereby cellular hypertrophy occurs initially in the upper anteroseptal wall and produces abnormal Q waves in the inferior and lateral leads even when wall hypertrophy is not detected by echocardiography.6Further studies are necessary to resolve the pathogenesis of abnormal Q waves in preclinical carriers of HCM.

4.3. Study limitations
Because the 148 subjects were from only 15 different families, the factors determining the characteristics of the subjects may not be independent from each other in performing statistical analysis. Furthermore, because this study did not include all disease-causing genes, it may be difficult to apply our results to all patients with HCM. Specifically, 47 of 92 genetically affected subjects (51%) had a Lys183del mutation in the cardiac troponin I gene. There may therefore be a significant bias in the subjects being studied. Finally, our results are applicable only in screening genetically affected subjects in familial HCM. It may be difficult to apply our results in subjects without a family history of HCM because the prevalence of HCM has been estimated to occur in less than 0.2% of the population.

5. Conclusions

This study is the first attempt to define the most accurate diagnostic criterion of abnormal Q waves based on a molecular genetic diagnosis. We propose that Criterion 3 (Q wave >3mm in depth and/or >0.04s in duration in at least two leads except aVR) may be the most accurate diagnostic definition for HCM. This study also found that abnormal Q waves may be one of the useful methods to identify preclinical carriers in clinically healthy subjects. Even in the post-molecular era, understanding the diagnostic value of abnormal Q waves in HCM may be of significant importance in screening carriers, especially those without left ventricular wall hypertrophy.

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