- Email: [email protected]
Analysis of ventricular myosin light chain genes in cardiomyopathy
Forensic Science International: Genetics Supplement Series 3 (2011) e143–e144
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Analysis of ventricular myosin light chain genes in cardiomyopathy Wataru Irie *, Chikako Murakami, Shigeki Nakamura, Kazuho Maeda, Chizuko Sasaki, Masataka Furukawa, Katsuyoshi Kurihara Department of Legal Medicine, Kitasato University School of Medicine, Sagamihara, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 August 2011 Accepted 29 August 2011
Recently, numerous mutations in sarcomeric genes are reported as factor of cardiomyopathy. Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are general disease in cardiomyopathy and causes sudden death in young people. In this study, ventricular myosin regulatory light chain gene (MYL2) and ventricular myosin essential light chain gene (MYL3) were analyzed in order to elucidate the cause of cardiomyopathy. In MYL3, a significant missense mutation (Ala57Gly) was detected in HCM case. This mutation was located in the EF-hand domain calcium-binding site that regulates the cardiac contraction. No genetic mutation was found in our previous report except this gene, it indicated that the mutation of the MYL3 was the main factor for the HCM case. It is necessary to survey the other disease causing genes to clarify the relationship with etiology. ß 2011 Published by Elsevier Ireland Ltd.
Keywords: Cardiomyopathy Sarcomeric genes Cardiac sudden death EF-hand domain Haplotype
1. Introduction Cardiomyopathy is defined as a heart disorder with cardiac dysfunction and is known to cause the sudden death. Since molecular genetic studies have identified several genes encoding sarcomeric proteins, genetic heterogeneity was considered as the principal cause of disease and we also investigated some popular genes mutation and reported previously [1–3]. Myosin is a molecular motor that is responsible for muscle contraction and is composed by two myosin heavy chain and two pairs of myosin light chain. The light chains are classified into essential light chain (ELC) and regulatory light chain (RLC). These proteins have an important function such as stability of myosin head and regulation of cardiac contraction by phosphorylation and Ca2+ binding [4]. In this study, we focused on MYL2 and MYL3, which codes RLC and ELC, respectively, in order to elucidate the cause of cardiac sudden death caused by cardiomyopathy. 2. Materials and methods DNA was extracted by QuickGene-800 (FUJIFILM) from blood specimens obtained from the cases of 15 HCM, 18 DCM and 3 ARVC (Arrhythmogenic right ventricular cardiomyopathy) with informed consent of their family members. The 200 cases that presented with no particular disease were used as control. Primers were designed for PCR amplification reactions necessary to cover
* Corresponding author at: 1-15-1, Kitasato, Minami-ku, Sagamihara-shi, Kanagawa 252-0374, Japan. Tel.: +81 42 778 9026; fax: +81 42 778 9026. E-mail address: [email protected] (W. Irie). 1875-1768/$ – see front matter ß 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.fsigss.2011.08.072
all coding exons of MYL2 and MYL3 (Table 1). Sequence analysis was essentially the same as those described previously [2]. This study has been reviewed and approved by the Hospital Ethics Committee of Kitasato University School of Medicine (B01-24). 3. Results and discussions Mutation screening of MYL2 and MYL3 identified silent mutation (Tyr152Tyr) and missense mutation (Ala57Gly), respectively (Fig. 1). The mutation in MYL3 was present in about 6.7% of examined HCM cases. The Ala57Gly was found in the case of 26-year old man. He was diagnosed as HCM one year ago and his brother also diagnosed as HCM. Autopsy was carried out and the diagnosis of HCM was confirmed. This mutation is known and that located in the EF-hand domain of the calcium-binding site, where the alignment of amino acid across the species and isoforms are highly conserved in ELC [4,5]. ELC plays an important role in the regulation of the contractile cardiac system, therefore the decreasing force generation was a primary mechanism for the pathogenesis of HCM associated with Ala57Gly. Also, there were no mutations in other sarcomeric genes investigated in our laboratory. The Tyr152Tyr that detected in MYL2 was a unique mutation. Gene frequencies and odds ratio of polymorphisms were calculated, no significant difference between cardiomyopathies and controls were detected. Although interestingly, polymorphisms detected in MYL2 exon3, intervening sequences (IVS) 4 and IVS5 performed haplotype. The haplotype in MYL2 and single nucleotide polymorphisms (SNPs) in MYL3 seemed to be highly polymorphic, so the examination of these regions might be applicable for DNA profiling. Multiple sarcomeric gene mutations were detected in some cardiomyopathy cases, it is necessary to analyze the
W. Irie et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e143–e144
e144
MYL2
EF-hand PhosphorylationCa2+ binding
Hinge region Myosin binding
NH2
COOH
12q23 2
1
3
4
5
6
Ile44Ile
7 Tyr152Tyr
IVS4 Ins GT [6521]
IVS5 DelG [7451] IVS5 InsC [7481] IVS5 -58 G>A
IVS4 +53 G>A
MYL3 Proline rich Actin binding
EF-hand domainEF-hand domain
EF-hand domain COOH
NH2
3p21 2
1 Pro23Pro
3 Ala57Gly
IVS1 +44 T>G
IVS3 +37 C>A
4
5 IVS5 C>T
-100
6
7 Exon7 G>A [6416]
Fig. 1. Schematic structure of sacomeric proteins and genes. Underline indicates polymorphism.
Table 1 The designed primers used for amplification of MYL2 and MYL3. Exon MYL2 1 2 3 4 5 and 6 7(1) (2) MYL3 1 2 3 4 5 6 7
Forward (50 –30 )
Reverse (50 –30 )
GTGCCCGCCTCACCTATGACT CACCCAGAGTAGGGGCCTGACCTA GGCCTGACGTTTTGAAGAAACTGT GGAGCCAATGGCAGTGTCATAATG AGCCTTGCTGGCCCCTTCATC
GGGGCATTGATTGGGGTTCCT TTCAGGCCGAATTTGGGATTGTTT TCCCATCCCCTGAATTTGTCTTA CGCCCAGCCAAGTTGCTGTTT TGTGGGGTCAGGGGTGCTTTAGAC
AGATGGCCGTGCCTTTATAC CCTGACGTGATGGCAACTT
TAACAGACAAGGTAGGGACAGAGG GAAACAGTTCTGAGTGGCTG
TTTATGGCCCTGTCCCTATTGTGC TGCTGGAATGGGACAGGCTGAGAC GGGCTGGGGTTCCCTGGTCTG AAAGTGCCTCGCGATGGTAGTTTG CCTGGCCTCAGAAATGGGGTGATG GAGGCTGACAGAAGACGAAGTGGA CTTGACATCCTGCTGCCTCC
CTCCTGCTGCAGCCCTTCTGATG ACATCCCCACACTGCCCGTAGGT CCCTCATCGGGACAATGCGAGAT GTCTGCCATTGAGGCTCCCTAATT ACCTGGGCACGAGGTTTAGCTGGAC AGACGGAGGCAGCAGGATGTCAAG AAAGAGAGGGAGGAAGGGAAAT
other sarcomeric genes and clarify the relationship with etiology. Conflict of interest statement None. References [1] J.G. Seidman, C. Seidman, The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms, Cell 104 (2001) 557–567. [2] C. Murakami, S. Nakamura, M. Kobayashi, Analysis of the sarcomere protein gene mutation on cardiomyopathy - mutations in the cardiac troponin I gene, Leg. Med. (Tokyo) 12 (2010) 280–283. [3] K. Maeda, S. Nakamura, C. Murakami, Analysis of three major sarcomeric genes (MYH7, TNNT2, MYBPC3) in cardiomyopathy, Forensic Sci. Int. Genet. Suppl. Ser. 2 (2009) 499–500. [4] I. Morgano, Tuning the human heart molecular motors by myosin light chains, J. Mol. Med. 77 (1999) 544–555. [5] W. Lee, T.H. Hwang, A. Kimura, Different expressivity of a ventricular essential myosin light chain gene Ala57Gly mutation in familial hypertrophic cardiomyopathy, Am. Heart J. 141 (2001) 184–189.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Analysis of ventricular myosin light chain genes in cardiomyopathy Wataru Irie *, Chikako Murakami, Shigeki Nakamura, Kazuho Maeda, Chizuko Sasaki, Masataka Furukawa, Katsuyoshi Kurihara Department of Legal Medicine, Kitasato University School of Medicine, Sagamihara, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 August 2011 Accepted 29 August 2011
Recently, numerous mutations in sarcomeric genes are reported as factor of cardiomyopathy. Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are general disease in cardiomyopathy and causes sudden death in young people. In this study, ventricular myosin regulatory light chain gene (MYL2) and ventricular myosin essential light chain gene (MYL3) were analyzed in order to elucidate the cause of cardiomyopathy. In MYL3, a significant missense mutation (Ala57Gly) was detected in HCM case. This mutation was located in the EF-hand domain calcium-binding site that regulates the cardiac contraction. No genetic mutation was found in our previous report except this gene, it indicated that the mutation of the MYL3 was the main factor for the HCM case. It is necessary to survey the other disease causing genes to clarify the relationship with etiology. ß 2011 Published by Elsevier Ireland Ltd.
Keywords: Cardiomyopathy Sarcomeric genes Cardiac sudden death EF-hand domain Haplotype
1. Introduction Cardiomyopathy is defined as a heart disorder with cardiac dysfunction and is known to cause the sudden death. Since molecular genetic studies have identified several genes encoding sarcomeric proteins, genetic heterogeneity was considered as the principal cause of disease and we also investigated some popular genes mutation and reported previously [1–3]. Myosin is a molecular motor that is responsible for muscle contraction and is composed by two myosin heavy chain and two pairs of myosin light chain. The light chains are classified into essential light chain (ELC) and regulatory light chain (RLC). These proteins have an important function such as stability of myosin head and regulation of cardiac contraction by phosphorylation and Ca2+ binding [4]. In this study, we focused on MYL2 and MYL3, which codes RLC and ELC, respectively, in order to elucidate the cause of cardiac sudden death caused by cardiomyopathy. 2. Materials and methods DNA was extracted by QuickGene-800 (FUJIFILM) from blood specimens obtained from the cases of 15 HCM, 18 DCM and 3 ARVC (Arrhythmogenic right ventricular cardiomyopathy) with informed consent of their family members. The 200 cases that presented with no particular disease were used as control. Primers were designed for PCR amplification reactions necessary to cover
* Corresponding author at: 1-15-1, Kitasato, Minami-ku, Sagamihara-shi, Kanagawa 252-0374, Japan. Tel.: +81 42 778 9026; fax: +81 42 778 9026. E-mail address: [email protected] (W. Irie). 1875-1768/$ – see front matter ß 2011 Published by Elsevier Ireland Ltd. doi:10.1016/j.fsigss.2011.08.072
all coding exons of MYL2 and MYL3 (Table 1). Sequence analysis was essentially the same as those described previously [2]. This study has been reviewed and approved by the Hospital Ethics Committee of Kitasato University School of Medicine (B01-24). 3. Results and discussions Mutation screening of MYL2 and MYL3 identified silent mutation (Tyr152Tyr) and missense mutation (Ala57Gly), respectively (Fig. 1). The mutation in MYL3 was present in about 6.7% of examined HCM cases. The Ala57Gly was found in the case of 26-year old man. He was diagnosed as HCM one year ago and his brother also diagnosed as HCM. Autopsy was carried out and the diagnosis of HCM was confirmed. This mutation is known and that located in the EF-hand domain of the calcium-binding site, where the alignment of amino acid across the species and isoforms are highly conserved in ELC [4,5]. ELC plays an important role in the regulation of the contractile cardiac system, therefore the decreasing force generation was a primary mechanism for the pathogenesis of HCM associated with Ala57Gly. Also, there were no mutations in other sarcomeric genes investigated in our laboratory. The Tyr152Tyr that detected in MYL2 was a unique mutation. Gene frequencies and odds ratio of polymorphisms were calculated, no significant difference between cardiomyopathies and controls were detected. Although interestingly, polymorphisms detected in MYL2 exon3, intervening sequences (IVS) 4 and IVS5 performed haplotype. The haplotype in MYL2 and single nucleotide polymorphisms (SNPs) in MYL3 seemed to be highly polymorphic, so the examination of these regions might be applicable for DNA profiling. Multiple sarcomeric gene mutations were detected in some cardiomyopathy cases, it is necessary to analyze the
W. Irie et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e143–e144
e144
MYL2
EF-hand PhosphorylationCa2+ binding
Hinge region Myosin binding
NH2
COOH
12q23 2
1
3
4
5
6
Ile44Ile
7 Tyr152Tyr
IVS4 Ins GT [6521]
IVS5 DelG [7451] IVS5 InsC [7481] IVS5 -58 G>A
IVS4 +53 G>A
MYL3 Proline rich Actin binding
EF-hand domainEF-hand domain
EF-hand domain COOH
NH2
3p21 2
1 Pro23Pro
3 Ala57Gly
IVS1 +44 T>G
IVS3 +37 C>A
4
5 IVS5 C>T
-100
6
7 Exon7 G>A [6416]
Fig. 1. Schematic structure of sacomeric proteins and genes. Underline indicates polymorphism.
Table 1 The designed primers used for amplification of MYL2 and MYL3. Exon MYL2 1 2 3 4 5 and 6 7(1) (2) MYL3 1 2 3 4 5 6 7
Forward (50 –30 )
Reverse (50 –30 )
GTGCCCGCCTCACCTATGACT CACCCAGAGTAGGGGCCTGACCTA GGCCTGACGTTTTGAAGAAACTGT GGAGCCAATGGCAGTGTCATAATG AGCCTTGCTGGCCCCTTCATC
GGGGCATTGATTGGGGTTCCT TTCAGGCCGAATTTGGGATTGTTT TCCCATCCCCTGAATTTGTCTTA CGCCCAGCCAAGTTGCTGTTT TGTGGGGTCAGGGGTGCTTTAGAC
AGATGGCCGTGCCTTTATAC CCTGACGTGATGGCAACTT
TAACAGACAAGGTAGGGACAGAGG GAAACAGTTCTGAGTGGCTG
TTTATGGCCCTGTCCCTATTGTGC TGCTGGAATGGGACAGGCTGAGAC GGGCTGGGGTTCCCTGGTCTG AAAGTGCCTCGCGATGGTAGTTTG CCTGGCCTCAGAAATGGGGTGATG GAGGCTGACAGAAGACGAAGTGGA CTTGACATCCTGCTGCCTCC
CTCCTGCTGCAGCCCTTCTGATG ACATCCCCACACTGCCCGTAGGT CCCTCATCGGGACAATGCGAGAT GTCTGCCATTGAGGCTCCCTAATT ACCTGGGCACGAGGTTTAGCTGGAC AGACGGAGGCAGCAGGATGTCAAG AAAGAGAGGGAGGAAGGGAAAT
other sarcomeric genes and clarify the relationship with etiology. Conflict of interest statement None. References [1] J.G. Seidman, C. Seidman, The genetic basis for cardiomyopathy: from mutation identification to mechanistic paradigms, Cell 104 (2001) 557–567. [2] C. Murakami, S. Nakamura, M.