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Postmortem molecular analysis to SIDS victims
Forensic Science International: Genetics Supplement Series 3 (2011) e263–e264
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Postmortem molecular analysis to SIDS victims M. Osawa *, Y. Inaoka, I. Hasegawa, F. Satoh Department of Forensic Medicine, Tokai University School of Medicine, Shimokasuya 143, Isehara, Kanagawa 259-1193, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 August 2011 Accepted 29 August 2011
In the present report, our recent molecular analyses to SIDS victims are introduced. The majority of congenital central hypoventilation syndrome patients carried the polyalanine repetitive expansion on exon 3 of PHOX2B. In contrast to the presence of the expansions in all clinically diagnosed CCHS patients, no abnormalities of the gene sequence were evident in a total of 87 SIDS cases. We finally concluded that CCHS should be too rare to be involved in SIDS. For long QT syndrome, a total of six non-synonymous substitutions were detected from direct sequencing of the responsible ion channel genes such as KCNQ1, KCNH2, and SCN5A in specimens from the victims. We therefore considered that SIDS might be partly explainable by fatal arrhythmia. Postmortem molecular analysis is not omnipotent in disease diagnosis, but this approach is effective for searching for disorders in which mainly physiological dysfunction is exhibited. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Congenital central hypoventilation syndrome Polyalanine repeat Long QT
1. Introduction
3. Results and discussion
It has been evident that dominant genetic disorders via de novo mutation at meiosis can be involved in various diseases. The sporadic occurrence of sudden, unexpected death from unknown causes could be attributable to heritable factors as well. Although conventional diagnostic examinations at postmortem were once limited to gross observation at autopsy, together with histology, molecular analysis has been developed as a novel approach to undiagnosed diseases ante-mortem. Here, we introduce our recent molecular analyses to the victims of sudden infant death syndrome (SIDS) concerning two genetic diseases of congenital central hypoventilation syndrome (CCHS) and long QT syndrome (LQT).
3.1. CCHS
2. Materials and methods DNA was prepared from intra-cardiac blood of SIDS victims obtained at autopsy as well as the peripheral blood of unrelated Japanese healthy volunteers and clinically diagnosed CCHS patients. Written informed consent was obtained from the subjects’ parents prior to experimentation. This study has been approved by the institutional ethical committee. Nucleotide substitutions were detected by direct sequencing, and the polyalanine tract was analyzed as previously described [1].
CCHS, also known as Ondine’s curse, is characterized by shallow breathing during sleep, with onset in the neonatal period. The gene responsible has been identified as PHOX2B, in particular the polyalanine expansion on exon 3 [2]. The repetitive tract and the vicinity exhibit high GC content (93%). To detect the polyalanine tract, tight secondary structure inhibited PCR reaction. To improve the amplification, we developed a modification using bisulfite treatment of DNA [1]. In the previous study, no mutations of the gene sequence were found in 48 SIDS cases, in contrast to the presence of the expansions in all clinically diagnosed CCHS patients. However, the symptom of apnea during sleep is quite similar to that of SIDS victims. We had thought that CCHS should be involved even in a small number of cases. After the last investigation, we continued to gather additional specimens of sudden infant deaths. In the present molecular analysis to the 33 additional cases, no polyalanine expansions, no non-synonymous substitutions and no indels in the gene were found once again, as summarized in Table 1. Finally, we concluded that CCHS should be too rare to be detected, or that the CCHS baby received clinical support after the mother noticed apnea. 3.2. LQT
* Corresponding author. Tel.: +81 463 93 1121; fax: +81 463 92 0284. E-mail address: [email protected] (M. Osawa). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.128
Long QT syndrome is occasionally fatal through syncope with critical arrhythmia, called torsades de pointes. Several responsible
e264
M. Osawa et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e263–e264
Table 1 Detected chromosome number (allele frequency) of PHOX2B variants in subjects of CCHS and SIDS groups.
Clinically diagnosed CCHS patients (n = 10) SIDS victims (n = 81) Healthy adults for the control (n = 190) a
Normal
Variantsa
10 (0.5) 162 (1.0) 380 (1.0)
10 (0.5) Not detected Not detected
not been carried out, we considered that several percentage of SIDS might be explainable by fatal arrhythmia associated with LQT [4]. Postmortem molecular analysis is not omnipotent in disease diagnosis, because many diseases are polygenic and are affected by acquired circumstances. However, this approach is effective for searching for a number of disorders in which mainly physiological dysfunction is exhibited.
Variants include the repeat expansions (n = 9) and deletion (n = 1) [1].
Conflict of interest genes, including ion channel genes such as KCNQ1, KCNH2, and SCN5A, have been identified. In the last investigation [3], a total of four non-synonymous substitutions were detected from direct sequencing of these genes in specimens from the last 42 SIDS victims. The recombinanat polypeptide molecules in oocytes exhibited the affected voltage dependence of activation. In the present study, the 33 additional specimens were analyzed by direct sequencing. As far as non-polymorphic sites, two nonsynonymous substitutions, R975Q (exon 17) and G1084S (exon 18), were detected in the SCN5A gene. Although the electrophysiological experiments using recombinant protein molecules have
None. References [1] H. Horiuchi, A. Sasaki, M. Osawa, et al., Sensitive detection of polyalanine expansions in PHOX2B by PCR using bisulfite-converted DNA, J. Mol. Diagn. 7 (2005) 638–640. [2] J. Amiel, B. Laudier, T. Attie-Bitach, et al., Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome, Nat. Genet. 33 (2003) 459–461. [3] T. Otagiri, K. Kijima, M. Osawa, et al., Cardiac ion channel gene mutations in sudden infant death syndrome, Pediatr. Res. 64 (2008) 482–487. [4] S.H. Opdal, T.O. Rognum, Gene variants predisposing to SIDS: current knowledge, Forensic Sci. Med. Pathol. 7 (2011) 26–36.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Postmortem molecular analysis to SIDS victims M. Osawa *, Y. Inaoka, I. Hasegawa, F. Satoh Department of Forensic Medicine, Tokai University School of Medicine, Shimokasuya 143, Isehara, Kanagawa 259-1193, Japan
A R T I C L E I N F O
A B S T R A C T
Article history: Received 29 August 2011 Accepted 29 August 2011
In the present report, our recent molecular analyses to SIDS victims are introduced. The majority of congenital central hypoventilation syndrome patients carried the polyalanine repetitive expansion on exon 3 of PHOX2B. In contrast to the presence of the expansions in all clinically diagnosed CCHS patients, no abnormalities of the gene sequence were evident in a total of 87 SIDS cases. We finally concluded that CCHS should be too rare to be involved in SIDS. For long QT syndrome, a total of six non-synonymous substitutions were detected from direct sequencing of the responsible ion channel genes such as KCNQ1, KCNH2, and SCN5A in specimens from the victims. We therefore considered that SIDS might be partly explainable by fatal arrhythmia. Postmortem molecular analysis is not omnipotent in disease diagnosis, but this approach is effective for searching for disorders in which mainly physiological dysfunction is exhibited. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Congenital central hypoventilation syndrome Polyalanine repeat Long QT
1. Introduction
3. Results and discussion
It has been evident that dominant genetic disorders via de novo mutation at meiosis can be involved in various diseases. The sporadic occurrence of sudden, unexpected death from unknown causes could be attributable to heritable factors as well. Although conventional diagnostic examinations at postmortem were once limited to gross observation at autopsy, together with histology, molecular analysis has been developed as a novel approach to undiagnosed diseases ante-mortem. Here, we introduce our recent molecular analyses to the victims of sudden infant death syndrome (SIDS) concerning two genetic diseases of congenital central hypoventilation syndrome (CCHS) and long QT syndrome (LQT).
3.1. CCHS
2. Materials and methods DNA was prepared from intra-cardiac blood of SIDS victims obtained at autopsy as well as the peripheral blood of unrelated Japanese healthy volunteers and clinically diagnosed CCHS patients. Written informed consent was obtained from the subjects’ parents prior to experimentation. This study has been approved by the institutional ethical committee. Nucleotide substitutions were detected by direct sequencing, and the polyalanine tract was analyzed as previously described [1].
CCHS, also known as Ondine’s curse, is characterized by shallow breathing during sleep, with onset in the neonatal period. The gene responsible has been identified as PHOX2B, in particular the polyalanine expansion on exon 3 [2]. The repetitive tract and the vicinity exhibit high GC content (93%). To detect the polyalanine tract, tight secondary structure inhibited PCR reaction. To improve the amplification, we developed a modification using bisulfite treatment of DNA [1]. In the previous study, no mutations of the gene sequence were found in 48 SIDS cases, in contrast to the presence of the expansions in all clinically diagnosed CCHS patients. However, the symptom of apnea during sleep is quite similar to that of SIDS victims. We had thought that CCHS should be involved even in a small number of cases. After the last investigation, we continued to gather additional specimens of sudden infant deaths. In the present molecular analysis to the 33 additional cases, no polyalanine expansions, no non-synonymous substitutions and no indels in the gene were found once again, as summarized in Table 1. Finally, we concluded that CCHS should be too rare to be detected, or that the CCHS baby received clinical support after the mother noticed apnea. 3.2. LQT
* Corresponding author. Tel.: +81 463 93 1121; fax: +81 463 92 0284. E-mail address: [email protected] (M. Osawa). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.08.128
Long QT syndrome is occasionally fatal through syncope with critical arrhythmia, called torsades de pointes. Several responsible
e264
M. Osawa et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e263–e264
Table 1 Detected chromosome number (allele frequency) of PHOX2B variants in subjects of CCHS and SIDS groups.
Clinically diagnosed CCHS patients (n = 10) SIDS victims (n = 81) Healthy adults for the control (n = 190) a
Normal
Variantsa
10 (0.5) 162 (1.0) 380 (1.0)
10 (0.5) Not detected Not detected
not been carried out, we considered that several percentage of SIDS might be explainable by fatal arrhythmia associated with LQT [4]. Postmortem molecular analysis is not omnipotent in disease diagnosis, because many diseases are polygenic and are affected by acquired circumstances. However, this approach is effective for searching for a number of disorders in which mainly physiological dysfunction is exhibited.
Variants include the repeat expansions (n = 9) and deletion (n = 1) [1].
Conflict of interest genes, including ion channel genes such as KCNQ1, KCNH2, and SCN5A, have been identified. In the last investigation [3], a total of four non-synonymous substitutions were detected from direct sequencing of these genes in specimens from the last 42 SIDS victims. The recombinanat polypeptide molecules in oocytes exhibited the affected voltage dependence of activation. In the present study, the 33 additional specimens were analyzed by direct sequencing. As far as non-polymorphic sites, two nonsynonymous substitutions, R975Q (exon 17) and G1084S (exon 18), were detected in the SCN5A gene. Although the electrophysiological experiments using recombinant protein molecules have
None. References [1] H. Horiuchi, A. Sasaki, M. Osawa, et al., Sensitive detection of polyalanine expansions in PHOX2B by PCR using bisulfite-converted DNA, J. Mol. Diagn. 7 (2005) 638–640. [2] J. Amiel, B. Laudier, T. Attie-Bitach, et al., Polyalanine expansion and frameshift mutations of the paired-like homeobox gene PHOX2B in congenital central hypoventilation syndrome, Nat. Genet. 33 (2003) 459–461. [3] T. Otagiri, K. Kijima, M. Osawa, et al., Cardiac ion channel gene mutations in sudden infant death syndrome, Pediatr. Res. 64 (2008) 482–487. [4] S.H. Opdal, T.O. Rognum, Gene variants predisposing to SIDS: current knowledge, Forensic Sci. Med. Pathol. 7 (2011) 26–36.