Chinese herb related molecules of cancer-cell-apoptosis: a minireview of progress between Kanglaite injection and related genes
2008

Yun Lu
Department of Hepatobiliary Surgery, Affiliated Hospital of Medical College
Qingdao University
No.16 Jiangsu Rd266003QingdaoPR China

Chang-Sheng Li
Center of Cell and Molecular Pathology, Affiliated Hospital of Medical College
Qingdao University
No.16 Jiangsu Rd266003QingdaoPR China

Qian Dong firstname.lastname@example.com
Department of Hepatobiliary Surgery, Affiliated Hospital of Medical College
Qingdao University
No.16 Jiangsu Rd266003QingdaoPR China

Department of Pediatric Surgery, Affiliated Hospital of Medical College
Qingdao University
No.16 Jiangsu Rd266003QingdaoPR China

Journal of Experimental & Clinical Cancer Research

Chinese herb related molecules of cancer-cell-apoptosis: a minireview of progress between Kanglaite injection and related genes

Journal of Experimental & Clinical Cancer Research
2731200810.1186/1756-9966-27-31Received: 22 June 2008 Accepted: 21 August 2008BioMed Central Page 1 of 5 (page number not for citation purposes) Review * Corresponding author This article is available from: http://www.jeccr.com/content/27/1/31
Many kinds of Chinese herb had been confirmed to have the character of anti-tumor, clinical reports about anti-tumor effects of Chinese herb had also been found in recent years, but most of the reports were focused on the clinical treatment of effectiveness for Chinese herb, on the other hand, review about Chinese herbal related with molecules on cancer-cell-apoptosis was seldom, many scientists could not believe such kinds of clinical describes about anti-tumor effects for Chinese herb, because these describes were lack of molecular biology evidence. Kanglaite(KLT) injection is an anti-tumor new drug which extracts from Chinese medicine-coix seed with modern advanced pharmaceutical technology, it is also a new biphase extended-spectrum anticancer medicine, the food and drug administration(FDA) of United States also approved a phase II trial of KLT to test its efficacy in treating non-small-cell lung cancer. Some studies show it could inhibit some anti-apoptotic gene and activate some pro-apoptotic gene, its injection solution is one of the new anticancer medicine that can significantly inhibit a various kinds of tumor cells, so it has become the core of research that how to further explore KLT injection to promote tumor cell apoptosis by impacting on related genes. In this review, the relationship between KLT and some tumor cell apoptosis molecules had been discussed and reviewed generally.

Review

In recent years, with the lucubrate on tumor cell biology and molecular biology, it has been recognized that the occurrence and development of tumor is not only the result of cell proliferation disorders and disdifferentiation, but also closely correlated with the abnormal apoptosis [1,2]. Although abnormal apoptosis can promote the occurrence and development of tumor, we can also treat tumor by promoting apoptosis of cancer cell [3,4]. Thus it has become the new target in oncotherapy by way of inducing apoptosis of cancer cell. Kanglaite(KLT) injectionis a diphasic broad-spectrum anti-tumor new drug which has depressant effect on many kinds of tumor cells, it is extracted from the chinese crude drug-coixenolide [5,6] and made use of the latest and most complex modern high technologies in process of preparation [7]. Animal experiments show that KLT mainly block G2+M phase of cell circle, thereby reducing the mitotic division of cells, so the proliferation of tumor cell was inhibited, at the same time it can also activate some pro-apoptotic factor, and further lead to apoptosis [8]. Clinical application also shows that combined with chemotherapy, KLT has a good effect on the treatment of advanced cancer, particularly in digestive tract cancer, for example, the patient's life span and quality of life improve significantly. The finding show that this preparation has significantly depressant effect and preonunced curative effect on a variety of cancer cells [9]. Although the therapeutic measure of liver cancer contain surgery, radiotherapy, chemotherapy, interventional therapy and so on, their effect is not satisfactory so far [10,11]. However, with the development of cell biology, the theory of apoptosis presents a new hope and path for the treatment of liver cancer, The present study has found that KLT plays an important role in promoting apoptosis of hepatoma cells [12]. It has been known that the apoptosis of hepatoma cells is triggered by a variety of receptor-mediated cell signaling, and a variety of protease take part in the apoptotic signal transduction. In addition many kinds of genes are also involved with apoptotic regulation of hepatoma cells. In this paper, the relationship between KLT and the hepatoma cell apoptosis molecules is going to be discussed and reviewed generally.

The influence of KLT on p53

There are two types of p53 genes: the wild type p53 gene and the mutant p53 gene. The wild type p53 gene, which is also known as the guardian of gene, is indispensable to regulate the normal cells circle [13,14]. On the one hand, as the important regulating factor in the process of apoptosis, the wild type p53 monitor the integrity of genes all the time, on the other hand, as the nuclear transcripton, it can respectively combines with DNA and RNA polymerase to regulate expression of gene, it can also inhibit the synthesis of DNA, take part in the repair of DNA, induce cell growth stop at the phase of G0. In addition, the wild type p53 gene can induce the Fas-mediated cell procedural death after the damage of DNA, so that the regular growth of cell is maintained. Wang JJ [8] had found, while discussing the anticancer mechanism of KLT injection, that the labeling index of wild type p53 in treat group which received KLT injection is 16.8%, while the control group had no expression at all. Furthermore, in the experiment about KLT-induced apoptosis, Bao Y [15] discovered that compared with the control group, the mRNA level of the wild type p53 gene significantly Increased in 20 μl/ml KLT experimental group after 48 hours. And in the experiment about apoptosis of multidrug resistance phenotypic human breast cancer cell line MCF7 adr and its cell cycle arrest that induced by KLT. According to the immunohistochemical detection, Guo JW [16] found that the wild type p53 gene of MCF7 adr in the control group is negative, but the wild type p53 gene of MCF7 adr in the KLT experimental group is midrange positive, it can be assumed that KLT could up-regulate the expression of p53 and extend half life of p53 protein. In addition, Wei CY [12] observed 34 cases' hepatoma carcinoma cell that cultivated in vitro and their changes after treated with KLT, the results indicated that, compared with the control group, the apoptosis of hepatoma carcinoma cell in KLT experimental group is very significant, there was significant difference between them, at the same time, the labeling index of wild type p53 in the treat group is (8.39 ± 1.42)%, but the labeling index of wild type p53 in the control group (2.11 ± 0.97)%, there was significant difference between these two groups. the phenomenon recorded here is the same as the studies of Wang JJ, Guo JW and Bao Y which had indicated [8,15,16]. In conclusion, KLT injection's may induce the apoptosis of tumor cell by way of up-regulate the expression of p53 genes.

The effect of KLT on the bcl-2 genes

Proto-oncogenes bcl-2 is the most definite apoptotic antagonist gene so far. In 1984, It was first cloned in t(14; 18) (q32; q21) chromosome translocation of follicular lymphoma cell line. But many studies had also confirmed that the high expression of this gene might inhibit the apoptosis of a variety of cells as well [17,18], thereby it can participate in the occurrence of a variety of tumor. When studying the anticancer mechanism of KLT, Wang JJ [8] found that the labeling index of bcl-2 gene was (16.80 ± 3.77)% in the control group, which is higher than that (6.6%) in the 10 μl/ml KLT treat group. Moreover, accompanied with the concentration of KLT increase, the gene expression of bcl-2 decreased in KLT group. In the experiment about KLT-induced the apoptosis of pancreatic cancer cells, according to the Western blot analysis, Bao Y [15] discovered that the expression of bcl-2 protein decreased after 72 hours when application of KLT at 20 μl/ml, the results of these two experiments above-mentioned may indicate that KLT induced the apoptosis of cells by downregulating the expression of bcl-2 genes. Nevertheless, when studying the KLT-induced the apoptosis of cancer cell(HL60), Li Y [19] make use of RT-PCR to detect the the gene expression of bcl-2, there was no significant change in genetic transcription after 24 hours when using KLT at 10 ul/ml. So, whether KLT induces apoptosis of cancer cell by down-regulating the expression of bcl-2 genes isn't yet clear, and its role in hepatoma is not learned, which requires further study and research.

The effect of KLT on Fas-genes

Fas gene is located on the No.10 chromosome q23, with a length about 25 kb, the codogenic Fas protein consists of 325 amino acids and it can be expressed in many tissues. When Fas protein combining with Fas ligand, signal of apoptosis is send to the cell and the apoptosis of cell is induce [20,21] Many anticancer drugs can induce the apoptosis of cancer cell by up-regulating the expression of Fas gene [22]. when studying the KLT-induced the apoptosis of cancer cell(HL60), Li Y [19] make use of RT-PCR to detect the the gene expression of Fas, she observed that genetic transcription strengthened after 24 hours when using KLT at 10 ul/ml. Han SX [23] proved that KLT injection can induce the apoptosis of human cervical carcinoma cell by raising the level of Fas gene. Similarly, the experiment, which detected the expression of Fas receptor on the surface of osteogenic sarcoma cell under different concentration of KLT [24], showed that under the KLT concentration of 0 ul/ml, 1 ul/ml, 5 ul/ml, 10 ul/ml and 20 ul/ml, the amount of Fas mRNA detected by the RT-PCR analisis is (0.12 ± 0.02) ul/ml, (0.27 ± 0.05) ul/ml, (0.35 ± 0.09) ul/ml, (0.46 ± 0.14) ul/ml and (0.51 ± 0.16) ul/ml respectively, so they considered that accompanied with the concentration of KLT increase, the level of Fas gene in the cancer cell significantly increased. Anyway, KLT may induce apoptosis of cancer cell by up-regulating the expression of Fas genes, but its effect on the Fas gene of liver cancer cell should be further studied.

The effect of KLT on caspase-3

Caspases is a group of prolease that induce the apoptosis of cell. Under the normal circumstances, the strict substrate specificity and high effectivity of the activated caspases can assures its narrow spectrum of proteolysis during the process of apoptosis [25,26]. The caspases selectively Shear a group of protein in a simpatico way that lead to functional failure or structural changes of the protein. If the activity of caspases is suppressed, the cellular apoptosis could be disturbed, which could lead to the occurrence and development of tumor, because the dynamic balance between cellular apoptosis and proliferation is disturbed. Caspases-3 and Caspases-8 are the most widely studied in caspases family. Caspase-3, as a important member in the caspases family, is a major functional enzyme in the pathway of cellular apoptotic signal [27,28]. Caspases-3 can cause the clearage of its substrate PARP(116 × 103) and transferred into opyeptide 24 × 103 and 89 × 103 and thereby activate the endonuclease to trigger the complete degradation of DNA. Many factors that regulate the cellular apoptosis can react through caspase-3 prolease. The depressor of caspase-3 can restrain the activation of caspase-3 and degrade the activity of it, so that the apoptosis of cancer cell is restrained [29,30]. Bcl-2 and p53 both interact with caspase family [31][32][33]. Some report have discovered, while studying KLT-induced the apoptosis of human pancreatic cancer Paru-8988 cell, that the increase of caspase-3 total protein also show obviously time dependence [34]. Moreover, through the test on the substrate of caspase-3-PARP, no degradation product (89 × 103) strap was found in the control group, but the degradation product (89 × 103) strap was discovered after 6 hours in KLT group, which indicates that caspase-3 have enzyme activity. However, no study or research about the effect of KLT on the liver cancer cell had done.

The effect of KLT on other related genes

PCNA (proliferating cell nuclear antigen) is a subunit of DNA polymerase and cell cycle-dependent protein whose maximum appearance in the S phase. Some research shows that the expression of PCNA is connection with the low grade tissues [35,36]. Wang JJ found that the gene expression of nuclear PCNA increased obviously, and the labeling index of PCNA was 15. 2% after 48 hours when 0.2 mg/ml KLT effected on the renal cancer cells, while hardly any gene expression of nuclear PCNA was observed in the control group [8]. They believed that PCNA took part in the reparative process of KLT-induced DNA injury, and when the degree of DNA injury is too serious to be repaired by PCNA, other genes such as P53 and Bcl-2 will send signals to trigger the apoptosis of cell and they were also convinced that the anticancer effect of KLT was a result of multiple gene interaction and restriction [8,15,16,19].

c-myc is one of the core protein -myc family in oncogene, and it not only is a positive controlling gene in cellular growth and cell life circle, but also take part in the progress of apoptosis, that is to say, it has dualism [37]. Under a circumstances of growth inhibiting, improper expression of c-myc could induce regulatory failure of normal cell life circle and apoptosis [38]. when studying the KLT-induced the apoptosis of cancer cell(HL60), Li Y make use of RT-PCR to detect the the gene expression of c-myc, she observed that there was no significant change in genetic transcription after 24 hours when using KLT at 10 ul/ml [19]. As bcl-2 gene, we should do further research and study on it. P21 WAFI/CIPI is the downstream gene of p53 gene, its activation contain the p53 dependent path and the p53 nondependent path [39]. The protein product of P21 WAFI/CIPI can combine with cell circle protein, cyclin-dependent kinase(CDK), and proliferating cell nuclear antigen(PCNA) to form a quaternionic complex that can stop the cell life circle and depress the cell growth. Guo JW discovered that while up-regulate the expression of p53 protein, KLT can raise the expression of p21 WAFI/CIPI mRNA and protein, it indicates that KLT can induce apoptosis of cancer cell by way of the p53 dependent path to up-regulate the expression of p21 WAFI/CIPI [16].

Furthermore, some researchers found that KLT can up-regulate the level of ubiquitin C, RAD17 genes and down regulate t the level of cyclin A, cyclin E1, cyclin F gene in studying the influence of KLT on Patu-8988 cell life circle and gene expression [40].

In addition, some study [41] show that the genes regulated apoptosis are not isolated, they can influence and restrict with each other, for example, Bcl-2 family, IAPs family, c-myc, P53, P35, can affect activation of caspase-3 through regulating activation of caspase-8 and caspase-9 [42]. so Kanglaite may promote the interaction of those genes to form a network cycle, amplify cascade reaction and further promote apoptosis. but there is no related research at the present stage.

Conclusion

Now, the study of KLT-induced apoptosis is focused on the above-mentioned gene. In short, KLT induce apoptosis of cancer cell by way of up regulating the expression of p53 gene, Fas gene, Caspase-3, PCNA, p21 WAFI/CIPI and down regulating the expression of cyclin A, cyclin E1, cyclin F gene. But its effect on the expression of bcl-2 gene and c-myc gene is not yet clear.

The occurrence of malignant tumor may be caused by the abnormal proliferation of cell or the inhibition of cellular apoptosis pathway. The proliferation of tumor cell and the apoptosis of tumor cell are not only affected by many factors and pathways such as drugs, radioactive ray, etc, but also regulated by some tumor genes or tumor-suppressing gene. It has been proved that the Chinese crude drug-induced apoptosis is one important anticancer mechanism of Chinese crude drug, and it is also relevant to the concentration of the medicine [43]. Just as the result of experimental treatment on hepatoma of mice that we have done [44], we found that KLT can made the cancer cells stop in the G2+M phase of cell life circle, and prevent them form entering the G0 and G1 phase. so it can induce the apoptosis and suppress growth of cells without any effect on the surrounding normal tissues or causing Inflammatory reaction which are unique characteristics. based on these characteristics, KLT has been used in the treatment on many kinds of malignant tumor, and the clinical effect show that KLT can not only repress tumor directly, improve the quality of life obviously, enhance the patient's immunity, but also enhance the effectiveness of chemotherapy and reduce side effects [45][46][47], which is consistent with its pharmacology above-mentioned However, the relationship between the apoptosis of hepatoma carcinoma cell and gene is further studied, although these research can not fully reveal the mechanism of KLTinduced apoptosis, and its experimental result is still preliminary, the trend of using KLT to prevent and cure liver cancer is already formed, and with the development of modern medical technologies, its mechanism will undoubtedly be revealed in the near future. and KLT will bring new hope for the treatment of cancer and the protection of normal tissues with the more sufficient evidence of its effect on liver cancer and other tumor in clinical trials.
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Competing interestsThe authors declare that they have no competing interests.Authors' contributionsLY wrote the article under the supervision of DQ. LY, LCS and DQ contributed to the collection and evaluation of date. LY conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.Publish with Bio Med Central and every scientist can read your work free of charge
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BMC Medical Genetics Adverse events in families with hypertrophic or dilated cardiomyopathy and mutations in the MYBPC3 gene

Philipp Ehlermann firstname.lastname@example.com
Dieter †1
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

Weichenhan email@example.com
Deutsches Krebsforschungszentrum
HeidelbergGermany

Jörg Zehelein
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

Henning Steen email@example.com
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

Regina Pribe email@example.com
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

Raphael Zeller firstname.lastname@example.com
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

Stephanie Lehrke firstname.lastname@example.com
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

Christian Zugck email@example.com
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

Boris T Ivandic
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

Hugo A Katus
Abt. Innere Medizin III
Medizinische Klinik
Universitätsklinikum Heidelberg
Germany

BMC Medical Genetics Adverse events in families with hypertrophic or dilated cardiomyopathy and mutations in the MYBPC3 gene
10.1186/1471-2350-9-95BioMed Central Page 1 of 11 (page number not for citation purposes) Research article * Corresponding author †Equal contributors
Background: Mutations in MYBPC3 encoding myosin binding protein C belong to the most frequent causes of hypertrophic cardiomyopathy (HCM) and may also lead to dilated cardiomyopathy (DCM). MYBPC3 mutations initially were considered to cause a benign form of HCM. The aim of this study was to examine the clinical outcome of patients and their relatives with 18 different MYBPC3 mutations.Methods: 87 patients with HCM and 71 patients with DCM were screened for MYBPC3 mutations by denaturing gradient gel electrophoresis and sequencing. Close relatives of mutation carriers were genotyped for the respective mutation. Relatives with mutation were then evaluated by echocardiography and magnetic resonance imaging. A detailed family history regarding adverse clinical events was recorded.Results:In 16 HCM (18.4%) and two DCM (2.8%) index patients a mutation was detected. Seven mutations were novel. Mutation carriers exhibited no additional mutations in genes MYH7, TNNT2, TNNI3, ACTC and TPM1. Including relatives of twelve families, a total number of 42 mutation carriers was identified of which eleven (26.2%) had at least one adverse event. Considering the twelve families and six single patients with mutations, 45 individuals with cardiomyopathy and nine with borderline phenotype were identified. Among the 45 patients, 23 (51.1%) suffered from an adverse event. In eleven patients of seven families an unexplained sudden death was reported at the age between 13 and 67 years. Stroke or a transient ischemic attack occurred in six patients of five families. At least one adverse event occurred in eleven of twelve families.Conclusion: MYBPC3 mutations can be associated with cardiac events such as progressive heart failure, stroke and sudden death even at younger age. Therefore, patients with MYBPC3 mutations require thorough clinical risk assessment.

Background

Hypertrophic cardiomyopathy (HCM) is a common disease with an estimated prevalence of 1:500 in north americans [1]. The disease may remain without clinical symptoms for many years. HCM has been recognized as the most common cause of sudden cardiac death in the young, especially in competitive athletes, where it accounts for 26% of cases [2]. Therefore, early diagnosis is crucial for prevention of such catastrophic events. For risk stratification, an expert based recommendation exists, which is mainly based on clinical findings such as maximum wall thickness [3], abnormal blood pressure response during exercise and non-sustained ventricular tachycardia [4]. Additionally, a positive family history for sudden cardiac death as a correlate of malignant gene mutations was recognized as an independent risk factor. Most cases of HCM are a result of mutations in genes encoding the sarcomeric proteins β-myosin heavy chain (MYH7), myosin binding protein C (MYBPC3), troponin T (TNNT2), troponin I (TNNI3), cardiac α-actin (ACTC) and α-tropomyosin (TPM1). Certain mutations in MYH7 and TNNT2 were ascribed a poor disease prognosis [5], while MYBPC3 mutations were attributed a more benign course and a low disease penetrance [6,7].

Dilated cardiomyopathy (DCM) is a major cause of heart failure in the young and accounts for about half of all heart transplantations. It is estimated that up to 35% of DCM cases are familial, indicative of potential gene mutations [8]. While HCM is mostly caused by mutations in sarcomeric protein genes, the genetic causes of familial DCM are more heterogeneous and hence, are less well defined. Up to 10% of familial DCM cases may be the result of mutations in sarcomeric protein genes like MYH7, MYBPC3 and TNNT2 [9]. Besides these genes, many others encoding cytoskeletal and nuclear envelope proteins, channel proteins and those encoding factors involved in calcium cycling were identified as candidates for DCM (for review [10]).

Here we studied the clinical outcome of families with HCM or DCM and mutations in MYBPC3, particularly with respect to adverse events like progressive heart failure, sudden cardiac death and stroke.

Methods

Patients

Patients presenting at the catheterization laboratory, cardiac MRI or outpatient department of our university hospital with a new or previously established diagnosis of a primary HCM or DCM were recruited for the study after written consent. Patients from external cardiologists were included, if a written consent and a blood sample was available. The diagnosis of primary HCM or DCM was established according to international standards. In short, HCM was defined by left ventricular wall thickening of 13 mm or more in the absence of secondary causes [11]. DCM was defined as depressed left ventricular ejection fraction less than 45% and a left ventricular end-diastolic diameter (LVEDD) of more than 117% of the expected value [12]. Echocardiography was performed in all patients according national and international standards. LVEDD was obtained from M-Mode recordings, maximum wall thickness and left ventricular ejection fraction (LVEF) form two dimensional echocardiography. LVEF was classified as follows: Normal ≥ 55%, mildly impaired 45-54%, moderately 30-44% and severely < 30% [13]. The study protocol was approved by the ethics committee of the medical faculty at the University of Heidelberg.

Cardiac MRI

After exclusion of contraindications, cardiac MRI was performed in supine position at end-expiration employing an 1.5 Tesla, whole body superconducting MRI scanner system (ACHIEVA, Philips Medical Systems, Best, the Netherlands) using a five-element cardiac phased array surface coil with vector ECG-gating. Assessment of resting LV function was determined by cine images using a steady-state free precession sequence (matrix = 160/256, sense-factor = 2, flip-angle = 60°, slice thickness/gap = 8/ 2 mm) in continuous short axes covering the whole left and right ventricle from base to apex as well as 2-, 3-and 4-chamber views in anatomically correct heart axes. MRI data was transferred to a clinical workstation (Viewforum, Philips Medical Systems, Best, the Netherlands). Enddiastolic (EDV, ml) and -systolic (ESV, ml) volumes and resulting ejection fraction (EF in %) were measured manually using short axes volumetry by determination of enddiastolic and -systolic cine images and subsequent delineation of endocardial borders excluding papillary muscles. For evaluation of myocardial mass, borders were also drawn at the interface between myo-and epicardium on end-diastolic images including papillary muscles. For anatomical description of late enhancement localisation, the AHA 17 segment model was employed consisting of six basal, six mid-ventricular, four apical segments as well as the apex. The signal intensities of late enhancement were described qualitatively (massive, normal, discrete) by consensus decision of the two observers.

Mutation screening 87 patients with HCM and 71 patients with DCM were screened by denaturing gradient gel electrophoresis (DGGE) and subsequent sequencing for mutations in coding exons and flanking intron sequences of the MYBPC3 gene as described previously [14]. In an experiment of our previous study [14] testing the sensitivity of DGGE, 16 of 16 sequence verified variants among 187 different patient samples were accurately identified, indicating the excellent sensitivity of the method. All patients with mutations in MYBPC3 were additionally screened for mutations by single-strand conformational polymorphism analysis as already described [15] in cardiac troponin I (TNNI3; exon 8 screened by direct sequencing) or by DGGE in β-myosin heavy chain (MYH7; with the exception of exon 27), cardiac troponin T (TNNT2), cardiac α-actin (ACTC) and α-tropomyosin (TPM1; with the exception of exon 1). The primer sequences used in this study are available on request. To examine whether newly identified MYBPC3 mutations are present in "healthy" individuals and, therefore, to be considered as polymorphisms rather than as disease-causing mutations, 430 control persons with normal systolic and diastolic function were screened for these new mutations by DGGE. These controls were patients recruited from our cardiac catheterization laboratory, had no significant stenoses revealed by coronary angiography and no evidence for any myocardial disease

Family screening

All first and second degree relatives of patients with a MYBPC3 mutation were asked to participate in our family screening program. Screening always included ecg and echocardiography. If an examination had been already performed by an external cardiologist, patients were asked for permission to request the examination record from their physicians. Furthermore, all relatives were invited to participate in the genetic screening and, if screened positive for a mutation, for additional cardiac MRI. This was performed in 16 of 42 mutation carriers. If possible, a pedigree of up to four generations together with a detailed family history was recorded. Deceased family members were considered affected if a sudden unexplained death occurred at or below 35 years of age [12]. Affection was presumed if a family member suffered from sudden death at 50 years of age or younger. Genetic counselling was offered to all participants and their families.

Results

MYBPC3 mutations in HCM and DCM patients

18 different mutations (four splice, three frameshift, seven missense, three nonsense mutations and one inframe codon deletion), distributed throughout almost the entire gene, from intron 2 to exon 34, were found in 18 patients (Tables 1 and 2, Fig. 1). Close relatives of mutation carriers were subsequently genotyped for the respective mutation. Seven mutations were considered novel, since they were neither found in the large MYBPC3 mutation list of the "Cardiogenomics" database http://genet ics.med.harvard.edu/~seidman/cg3/muts/ MYBPC3_mutations_TOC.html, nor in the SNP database dbSNP http://www.ncbi.nlm.nih.gov/projects/SNP/, nor in one of recent articles found in PubMed http:// www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed describing novel mutations in the MYBPC3 gene. The causal role of the new mutations in the disease was underscored by their absence in 430 control individuals with normal systolic and diastolic function and, in one case (family 16), familial co-segregation with the disease. Moreover, four of the seven new mutations lead to a significant change of the amino acid sequence, namely two splice site mutations, one frameshift mutation and one nonsense mutation. A fifth mutation, p.R272C, altered one of three known phoshorylation motifs of the MYBP-C protein [16]. Preliminary analyses revealed a threefold decrease in the phosphorylation of a mutant p-R272C MYBP-C peptide fragment compared to the wild type fragment (unpublished data), yet the functional consequence of the mutation in vivo is still unknown. The two remaining missense mutations are considered functionally relevant since both affect highly conserved amino acid positions (Fig.  2).

Two new mutations were found in DCM patients of families 5 and 16, while all other mutations were detected in patients with HCM. One patient was a double-heterozygote for two different missense mutations located in exons 7 and 28. Members of the two unrelated families 17 and 18 had the same Gln1233ter nonsense mutation, which terminates the MYBP-C protein prematurely, leading to loss of 42 of the 1274 amino acids. This mutation had been described previously as a polymorphism [17], based on its presence in 2% of 200 phenotypically uncharacterized control individuals. We and others, however, considered the mutation disease-causing, since it was neither detected in 350 controls in previous studies [18,19], nor in our 430 controls. Moreover, the mutation clearly segregated with the disease in all five mutation carriers of family 17, either associated with a HCM phenotype or with borderline myocardial hypertrophy (IV-2).

To exclude mutations in other genes of high relevance in cardiomyopathies, the 18 initial mutation carriers were screened additionally for mutations in MYH7, TNNT2, TNNI3, ACTC and TPM1 encoding β-myosin heavy chain, cardiac troponin T, cardiac troponin I, cardiac α-actin and α-tropomyosin, respectively. No further mutations were found in these genes.

Phenotypic heterogeneity of mutation carriers

We observed a broad spectrum of phenotypes among the mutation carriers: from asymptomatic and phenotypically normal at the age of 85, like I-2 of family 5 (Table 2), to heavily affected at a relatively young age, like III-9 of family 16 at the age of 44 or IV-3 of family 12 at the age of 13 (Tables 2 and 3). Even close relatives carrying the same mutation displayed a striking phenotypic heterogeneity, as documented by members of families 5, 12, 16 and 17.

The index patient of family 5, II-2, initially presented with decompensated heart failure during new onset of tachyarrhythmic atrial fibrillation and arterial hypertension. In addition, cardiac catheterization ruled out significant coronary artery disease, but revealed an enlarged left ventricle with severely depressed systolic function. The patient improved, however, under guideline-conform treatment, as documented four years later by cardiac MRI displaying normalized left ventricular function and only minor pathological findings regarding wall thickness and myocardial late contrast enhancement. The son, III-2, had a mild enlargement of the left ventricle with an end-diastolic volume index of 98.1 ml/m 2 during MRI (reference value 47-92 ml/m 2 ). However, he and his father showed a marked trabeculation on cardiac MRI. This finding did not fulfil the criteria for myocardial noncompaction, but might be an explanation for the overestimated wall thickness by echocardiography. Two further mutation carriers of family 5, I-2 and II-6, were asymptomatic and, as revealed by cardiac MRI, showed no phenotypical abnormalities.  Pedigrees of affected families: Open and black symbols represent unaffected and affected individuals, respectively  The index patient of family 12 (III-3) had a severe left ventricular hypertrophy with outflow obstruction, which required septal ablation. His son (IV-3), who suffered from sudden cardiac death at the age of 13 years, also had a severe hypertrophy. In contrast, the index patient's mother (II-6), also mutation carrier, had only a borderline hypertrophy. In this patient, a comorbidity with a coronary heart disease and previous inferior infarction was prevalent, and the ventricular arrhythmias found at holter monitoring might be a result from a myocardial scar after inferior infarction.

In family 17, the index patient, II-3, presented at the age of 72 years with a leading finding of a restrictive pathophysiology and only a mild wall thickening, which probably was a result of an end-stage HCM. Unfortunately, high-quality cardiac MRI recording was impossible due to atrial fibrillation. All three children of this patient were mutation carriers; only one was previously diagnosed with HCM, while the other son and the daughter were diagnosed with HCM in the course of our family screening.

Adverse clinical events

Malignant clinical events such as unexplained sudden death, stroke or transient ischemic attack were ascertained in many members of our study families (Table 3). Based on twelve families and six single patients, a total number of 42 mutation carriers was identified, of which eleven had at least one adverse event. A total number of any adverse event occured in 23 of 45 (51.1%) cardiomyopathy cases. Eleven individuals of seven families suffered from sudden death between 13 and 67 years of age. In three families, a sudden death occurred at or before the age of 35 years. Any adverse event occurred in eleven of twelve families and in two of six single mutation carriers. The youngest, IV-3 of family 12, already diagnosed with an extreme septal hypertrophy and a dynamic outflow obstruction, died at the age of 13. Moreover, a first degree relative, II-3, of a mutation carrier in the same family experienced sudden death at the age of 33. In family 16, a sudden death occurred only shortly after the diagnosis of HCM had been ascertained in the course of family screening. The female patient, III-9, had no heart failure symptoms but a maximum wall thickness of 22 mm, moderate late enhancement during contrast MRI and mild elevated NT-proBNP. Clinical findings in the patient did not qualify for an implantable cardioverter defibrillator (ICD) based on available risk scores [4]. A formerly asymptomatic individual, II-1, of the same family suffered from sudden cardiac death at the age of 32 in relation to exercise. This patient had not been genotyped, yet can be considered mutation carrier based on the fact that his two brothers, II-3 and II-5, and two symptomatic children, III-3 and III-6, are mutation carriers.

Five patients of four families received an ICD for primary prevention because of non-sustained ventricular tachycardia or other high-risk disease profiles. Stroke or a transient ischemic attack related to atrial fibrillation occurred in six patients of five families between 51 and 64 years of age. Two of these patients had persisting disabling symptoms. Two patients with stroke and two with a transient ischemic attack recovered without permanent impairment.

Genotype-phenotype correlation

Sudden death as the most serious adverse event was prevalent in seven of 18 families (39%). However, despite missense mutations were most frequent with seven of 18 index cases, none of the sudden deaths was observed in families of these patients. In contrast, sudden deaths occurred in three of four families with splice mutations, in two of three with nonsense mutations, in one of three with frameshift mutations and in the single family with an in-frame codon deletion. All three families with sudden deaths at or before the age of 35 years were carriers of splice mutations.

Discussion

Mutations in the MYBPC3 gene are among the most prevalent causes of inherited HCM. In earlier studies, e.g. [6,7], MYBPC3 mutations were found to be associated with a low penetrance, late onset and a benign course of the disease. This notion became challenged, however, by the findings of two recent studies. In the first, carriers of a specific MYBPC3 frameshift mutation from 15 unrelated Conservation of mutated MYBPC3 amino acid residues throughout different species Figure 2 Conservation of mutated MYBPC3 amino acid residues throughout different species.

I336V T957S

Human SEYERIAFQYG PGAPVTTTEPV Dog SEYERIAFQHG PGAPVTTQEPV Cattle SEYERIAFQHG AGPPVTTKEPV Claw frog SEYEKIAFQYG PSEPCTMKEPV Zebrafish SEYEKIAFQYG PSAPATLQQAV , showed also borderline signs of the disease. Only three mutation carriers, all members of family 5, had no certain pathological findings. The corresponding mutation, a novel p.R272C missense mutation, altered one of three known phoshorylation motifs of the MYBP-C protein [16]. We found decreased phosphorylation at this position in vitro, but we don't know the functional consequences in vivo. In this family, only the index patient showed a DCM phenotype, for which a different cause, e.g. hypertensive heart disease, cannot be ruled out. The other family with a DCM phenotype of the index patient was family 16 with a novel splice site mutation. Two older mutation carriers suffered from DCM, while four younger patients from the following generation either showed symptoms of HCM or at least a HCM phenotype upon cardiac MRI examination. It is most likely that the two older patients suffered from an end-stage HCM with progression to a DCM phenotype, as had been described already in a few cases of MYH7 and MYBPC3 mutation carriers [22]. For still unknown reasons, the number of MYBPC3 mutations associated with DCM is much lower [23] than the number of MYBPC3 mutations associated with HCM.

Contrasting to previous findings in families with MYBPC3 mutations [7], malignant clinical events were frequently observed in our families, even at a young age. Sudden cardiac deaths at or even before the age of 35 did occur in four members of families 6, 12 and 16. In accordance with current recommendations [4], these data underscore the importance of a close meshed risk stratification in every patient at risk due to a mutation in MYBPC3. Although the number of mutations is too small to prove statistically significant, it is remarkable that all sudden deaths at or before the age of 35 occurred in families with splice mutations, while no sudden death was observed in families with missense mutations. Stroke and peripheral embolizations had been reported by others in 6% of HCM patients, with an incidence of 0.8% per year [24]. As several factors in HCM are favouring manifestation of atrial fibrillation, it is not surprising that in five of 18 families a history of stroke or transient ischemic attack was prevalent. Therefore, clinical care of patients and family members at risk should consider risk indicators for atrial fibrillation like left atrial enlargement, severe diastolic dysfunction or family history of stroke or high susceptibility for atrial fibrillation.

A major limitation of our study is that genotype and phenotype data of deceased family members are not available in most cases. At a more advanced age, alternative causes of sudden death like myocardial infarction and atrial fibrillation become more likely. Therefore, only a well documented sudden death below 35 years of age is an accepted criterion for familial involvement in a cardiomyopathy [12]. In our retrospective evaluation of affected families, it cannot be excluded, that other conditions may have led to the adverse events. Nevertheless, it is noteworthy that in many of our families adverse events are prevalent in first-degree relatives at both older and younger ages, while MYBPC3 mutations initially were believed to be associated with a good prognosis. However, a referral bias in our observational study cannot be excluded, although the reason for presentation in virtually all index patients were diagnostic procedures to rule out concomitant coronary artery disease, but not adverse events. Mutations in other likely candidate genes for HCM were not found in any of the index patients. As to the heterogeneity of the genetic causes of cardiomyopathies, additional mutations in less likely or hitherto unknown disease genes cannot be excluded. Moreover, yet unknown disease modifiers might have had a modulating effect on the disease phenotype. The question of the prognostic impact of certain mutations in cardiomyopathies has to be clarified by inclusion of mutations carriers into registries.

funding, supervised the study and manuscript drafting. The manuscript was written and approved by all authors.

Figure 1
1Pedigrees of affected families: Open and black symbols represent unaffected and affected individuals, respectively. Dashed symbols indicate individuals with presumed or borderline disease phenotype. Grey symbols denote individuals with unknown clinical status. (+) and (-) symbols indicate presence and absence of the mutation, respectively. Index patients are marked by an arrow. Slanted bars denote deceased individuals.

16, the index patient, II-3, and his brother, II-5, had a DCM phenotype. In contrast, the other mutation carriers, all belonging to the third generation of family 16, displayed a HCM phenotype upon cardiac MRI examination, yet were otherwise asymptomatic. Only in individual III-3, a HCM was diagnosed before during a routine physical examination.

Table 1 :
1MYBPC3 mutations detected in this study No. of family or single patient Exon/IVS* Nucleotide position † and change Amino acid change Affected ‡ Genotyped total/ pos.Reference,
comment

Table 2 :
2Clinical characteristics of mutation carriersNo.* Indiv. Sex Age
Age at first
presentation
(Diagnosis)

ECG***
Echo
MRI

MWT  † (mm) LVEF  ‡ MWT  † (mm) LVEF (%) Late enhancement

1
II-3  §
m
56
49 (HCM)
SR, CV
28
n
27
60
mid-, inferoseptal and
inferior basal
II-9
m
55
53 (HCM)
AF, LBBB
30
n-↓
33
51
massive septal,
anterior, inferior
2
II-1  §
m
55
55 (HCM)
pxAF
24
n
III-1
f
30
30 || (HCM)
SR
15
n
3
SP #
m
71
67 (HCM)
AF
20
n-↓
4
SP #
m
48
46 (HCM)
SR, CV, iLBBB
25
↓

5
I-2
f
85
n.a. (-)
AF
11
n
II-2  §
m
65
62 (DCM)
SR, CV
15  † †
n
1 2 . 5
7 2  ‡ ‡
discrete septal
(mid and basal)
II-6
f
55
55 || (-)
SR
10
n
< 11
65
none
III-2
m
38
38 (susp.)
SR
14  † †
n
< 1 1
6 2  §S
none
6
II-2
f
68
n.a. (HCM)
SR
n.a.
III-1  §
m
39
18 (HCM)
SR
n.a.
7
SP #
f
52
45 (HCM)
SR, CV
n.a.
n
8
I -2
f
7 1
6 9 || (HCM)
SR
20
n
II-1  §
m
49
35 (HCM)
SR, AVB1, pxAF
25
↓
26
71
n.a.
9
SP #
f
66
44 (HCM)
SR, pxAF
24
n
10
SP #
m
54
54 (HCM)
SR
22
n
11
II-3
m
66
66 || (HCM)
AF
20
n
II-6  §
f
62
54 (HCM)
SR
22
n-↓
12
II-6
m
67
67 || (susp.)
SR, LAHB
12
n
III-1
m
46
42 || (HCM)
SR
14
n
21
54
massive intramural
basal-and mid-septal
III-3  §
m
42
37 (HCM)
SR
30
n
IV-4
f
20
20 || ( H C M )
S R
1 4
n
1 4
7 7
n o n e
IV-5
f
14
14 || ( s u s p . )
S R
1 3
n
1 1
6 6
n o n e
13
II-1
m
54
54 (HCM)
SR
13
n
II-6  §
m
42
21 (HCM)
SR
27
↓

14
II-2  §
f
61
57 (HCM)
SR
14 ||
n
II-5
m
55
55 || (HCM)
SR
18
n
20
74
mid and basal septal
15
SP #
m
49
49 (HCM)
SR
19
n
24
62
anteroseptal basal
16
II-3  §
m 66 ##
57 (DCM)
AF
n.a. ##
↓↓↓ ##

II-5
m
65
34 (DCM)
AF, LBBB
12
↓↓↓

III-3
m
42
18 (HCM)
SR
15
n
22
67
intense septal
III-6
f
40
40 || (HCM)
SR
15
n
III-7
f
39
39 || (HCM)
SR
15
n
14.5
72
septal, inferoseptal
basal
III-9
f
44
44 || (HCM)
SR, iLBBB
16
n
22
71
midseptal and
inferoseptal basal
17
II-3  §
m
72
n.a. (HCM)
AF, RBBB
16
n
III-6
m
48
n.a. (HCM)
SR
24
n
III-8
f
43
43 || (HCM)
SR
10
n
13.5
65
discrete basal-septal
III-9
m
42
42 || (HCM)
SR
18
n
IV-2
m
17
17 || (susp.)
SR
12
n
18
II-4  §
f
37
27 (HCM)
SR
23
n
24
66
massive intramural
basal-and mid-septal
III-5
m
15
15 || (susp.)
SR
12
n

*Number of family or single patient (SP);  † MWT: maximum wall thickness;  ‡ Left ventricular ejection fraction (n: normal; ↓: mildly reduced, ↓↓↓:
severely reduced);  § Index patient; || First presentation due to screening program; # single patient; **n.a.: not available;  † † marked trabeculation;  ‡ ‡ End
diastolic volume 205 ml, EDV index 94.9 ml/m 2 (reference value 47-92 ml/m 2 );  § § End diastolic volume 212 ml, EDV index 98.1 ml/m 2 ; || || 4 years after
transcoronary ablation of septal hypertrophy; ## heart transplantation with 58 years; ***ECG: electrocardiogram; SR: sinus rhythm; CV: status after
electrical cardioversion due to atrial fibrillation; AF: atrial fibrillation; pxAF: paroxysmal AF; LBBB: left bundle branch block; iLBBB: incomplete
LBBB; AVB1: first degree atrio-ventricular block; LAHB: left-anterior hemiblock; RBBB: right bundle branch block

Table 3 :
3Adverse events in families of MYBPC3 mutation carriers Here we screened 87 patients with HCM and 71 patients with DCM for mutations in MYBPC3 and correlated the genotypes, phenotypes and clinical outcomes of the mutation carriers and their close relatives. We identified 18 different mutations in 18 index patients, 16 with HCM and 2 with DCM. Mutations in other likely candidate genes for HCM and DCM were excluded. In addition to the 18 index patients, 24 more MYBPC3 mutation carriers were detected among relatives, distributed over ten families. In ten of them, the presence of a cardiomyopathy was unknown before. 16 of the 24 additional mutation carriers displayed a clear disease phenotype. Additional three individuals had a borderline phenotype: One of them had hypertension as potential cause of secondary hypertrophy and coronary artery disease. Two more and still young individuals, a 17 year old male (IV-2 of family 17) and a 15 year old female (III-5 of family18)Fam or single patient Patient
Confirmed mutation carrier
Age at event
Type of event

1
II-9
Yes
54
Transient ischemic attack

2
II-1
Yes
55
Stroke

II-4
No*
49
ICD  †

6
I-2
No*
35
Sudden death

7
Yes
51
Stroke

8
II-1
Yes
49
Pacemaker (Sick sinus syndrome)

9
Yes
59
Palpitations, positive EPS  ‡ , ICD  †

11
I-2
No*
55
Sudden death

II-2
No*
36
Sudden death

12
I-1
No*
42
Sudden death

II-3
No*
33
Sudden death

II-6
Yes
67
Inferior MI, coronary heart disease
67
ICD  † : Non-sustained VT (holter)

III-3
Yes
42
ICD  † : Non-sustained VT (holter)

IV-3
No*
13
Sudden death

13
I-1
No*
64
Stroke
67
Sudden death

II-1
No*
53
Transient ischemic attack

14
I-1
No*
53
Sudden death

16
II-1
No*
32
Sudden death

II-3
Yes
58
Heart transplantation

II-5
Yes
61
Resuscitation due to ventricular fibrillation, ICD  †
67
Heart failure related death

III-9
Yes
45
Sudden death

17
II-3
Yes
64
Stroke

18
I-2
No*
42
Sudden death

*First degree relative with mutation;  † ICD: implantable cardioverter defibrillator;  ‡ EPS: electro-physiological study (programmed ventricular
stimulation)

(page number not for citation purposes)
AcknowledgementsThe excellent technical assistance of Sandra Manthey and Oliver Mücke is gratefully acknowledged.Dieter Weichenhan acknowledges support by grant NGFN 01GS0420 (project no. NHK-S02T09) from the Bundesministerium für Bildung und Forschung.ConclusionThe finding of a MYBPC3 gene mutation is not generally associated with a good prognosis. Therefore, MYBPC3 mutation carriers should be as well subjected to careful clinical risk stratification.Competing interestsThe authors declare that they have no competing interests.PEAuthors' contributionsPre-publication historyThe pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2350/9/95/prepub
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