／【Ｋ：】Ｓｅｒｖｅｒ／１５０１０４７９ 新潟県厚生農業協同組合／本文／０４

五十嵐００１‐００４

2015.02.18 19.03.3

Genetic analysis of the myocardial Na+ pump α subunit gene SCN５A mutation in ７ cases of sudden
厚生連医誌
death among
第２４our巻 ４２５１号
autopsy
１〜４cases -２０gene
１
５mutation was suggested in４cases out of７ones by the polymerase chain reaction-single strand conformational polymorphism（PCR-SSCP）method.

Original article

Genetic analysis of the myocardial Na+ pump α subunit gene
SCN５A mutation in ７ cases of sudden death among our ４２５
autopsy cases - gene mutation was suggested in ４ cases outof ７ ones by the polymerase chain reaction-single strand
conformational polymorphism（PCR-SSCP）method.
Nagaoka Central General Hospital, Department of pathology ; Pathologist

Toshihiko Ikarashi
Introduction : Concerning the unknown sudden death inspite of the pathologic autopsy survey, we carriedout the genetic study for cardiac sudden death.
Materials and methods : Among ７ cases of sudden deathfrom ４２
５ autopsied cases in our institution for recent １
５ years, we examined myocardial Na+ pump
α subunit gene SCN５A mutation with the DNA
specimens derived from their formalin-fixed paraffin-embedded sections（FFPEs）by PCR-SSCP
method.
Results : Genetic mutation was shown in ４ cases out of ７ones, and they were correctly re-diagnosed as cardiac sudden death.
Conclusion : For the further investigation of the sudden
death, the genetic analysis is effective.

Brugada syndrome revealed SCN５A mutation in exons
１２, １８, or ２０（６）. We reviewed our unknown-suddendeath autopsied cases by SSCP analysis of these SCN５A
mutations.

Materials and methodsDNA samples were extracted from FFPE specimens with neutral formalin fixative and DEXPAT（Takara）（Table１）（４）.
４ pairs of PCR primers were used against exons
１２-１, １２-２, １８, and ２
０ of Na+ pump α subunit gene SCN
５A（Table２）（６）. PCR component and amplification setting were same as the previous one（Table ２）（３）. The
numbers of amplification cycles were from ３
０ to １２０, depending on producing appropriate each single optimum
electrophoretic band of PCR products. Mean PCR cycles
were３０ for exon １２-１, ４５ for exon １２-２, ８０ for exon １８,and １２０ for exon ２０. Because of many amplification cycles, the chemical reagent adjustment manipulation likenested PCR was done during the amplification.
SSCP method with silver stain was complied withthe previous report except several modifications : the useof half-sized electrophoresis device measuring １
８ cm inlength, and electrophoresis at ２８０V for ２ hours until bluestained presentation markers flowed out（Table２）（２）.
The criteria judging the aberrant band as positive was as follows :１. golden-colored bands similar asones of the control case, ２. definite black bands, confirmed by the reexamination.

Key words : genetic analysis, myocardial Na+ pump α
subunit gene SCN５A mutation, cardiac sudden
death, autopsy, gene mutation, polymerase chain
reaction-single strand conformational polymorphism（PCR-SSCP）method

Introduction
Sudden death consisted of many causes : cerebral,cardiac, aortic, anaphylactic, drug-abuse and so on. The
process of making final diagnosis has been improved byseveral new techniques : i.e. the autopsy imaging for aspace-occupying hematoma, the immuno-histochemical
study with anti-complement ９ antibody for an extremely
early phase of myocardial necrosis（５, ７）. A death causeof functional disturbances, like lethal arrhythmia and subsequent myocardial dysfunction, has been, nevertheless,left unsolved. Myocardial Na+ pump α subunit gene SCN
５A mutation has been identified as the cause of lethal arrhythmia（１, ６）. Gene SCN５A consisted of ２
８ exons, ８０kb in size. Ito found that ３３％ of arrhythmic cases ofResults
Final PCR products were around２００-３００bp.
Case ４ and ５ showed aberrant bands in exon １２-１（Figure１）. Case ６ and ７ revealed extra bands in exon―1―／【Ｋ：】Ｓｅｒｖｅｒ／１５０１０４７９ 新潟県厚生農業協同組合／本文／０４

厚生連医誌

第２４巻１号

五十嵐

００１‐００４

2015.02.18 19.03.3

１〜４ ２０１
５１８（Figure １）. Cardiac sudden death was suggested inthese four cases（Table１）.

able from : URL. http : //www.janiigata.sakura.ne.jp/
JMNK/１０-１/１６.pdf.
５．Ikarashi T. Advocacy of imaging diagnosis before
autopsy（Autopsy imaging, AI）. Niigata-Ken KoseirenMed J ２００３;１２:９
９-１０
２. Available from : URL. http ://www.janiigata.sakura.ne.jp/JMNK/１２-１/９９.pdf.
６．Itoh H. Clinical characteristics and SCN５A gene mutations in patients with Brugada syndrome. J Juzen Med
Soc ２００１;１１０:３４８-５
９. http : //dspace.lib.kanazawa-u.
ac.jp/dspace/bitstream/２２９
７/４
５６１/１/C００００００２２３
７-００８ito.pdf.
７．Piercecchi-Marti MD. et al. Immunostaining by complement C９: a tool for early diagnosis of myocardial
infarction and application in forensic medicine. J Forensic Sci２００１;４６:３２
８-３４.

Discussion
Cardiac sudden death was mainly caused by myocardial infarct, which was confirmed by coronary obstruction and myocardial necrosis. Extremely early phase of
myocardial infarct without typical myocardial necrosis became diagnosed by immune-histochemical positivity
against complement ９（７）. But there seemed to be many
cardiac sudden deaths arisen by lethal arrhythmia or myocardial dysfunction without any organic pathologicchanges. Myocardial Na+ pump α subunit gene SCN５A
mutation as the cause of lethal arrhythmia and myocardial
dysfunction can support these sudden cardiac death（１,６）.
In this study four autopsied cases with unknown sudden
death could be disclosed as cardiac sudden death based on
SCN５A mutation. The aberrant stains in SCN５A-SSCP
method were shown in４／７（５７％）, which was higher thanthat of the previous report（６）. These genetic analyses become effective in an accurate diagnosis of functional cardiac failure.

和 文 抄
原録

著

ナトリウムポンプ α サブユニット SCN５A 遺伝子変異を polymerase chain reaction-single strand conformational
polymorphism（PCR-SSCP）手技で同定できた心臓性突然死の４症例

References

長岡中央綜合病院、病理部；病理医い１．Aizawa Y et al. Guidelines for risks and preventionof sudden cardiac death（JCS２０１０）. Available from :URL. http : //www.j-circ.or.jp/guideline/pdf/JCS２０１０aizawa.h.pdf.
２．Hasegawa H., Ikarashi T. Detection analysis of thep５
３ genomic mutations by polymerase chain reactionsingle strand conformational polymorphism（PCRSSCP）method. Niigata-Ken Koseiren Med J２０００;１１:
４２-５. Available from : URL. http : //www.janiigata.
sakura.ne.jp/JMNK/１１-１/４
２.pdf.
３．Hasegawa H., Ikarashi T. Identification of mycobacterium tuberculosis DNA in formalin-fixed and paraffin-embedded tissue by the polymerase chain reaction. Niigata-Ken Koseiren Med J ２０００;１
０:２
１-４. Available
from : URL. http : //www.janiigata.sakura.ne.jp/JMNK/
１０-１/２
１.pdf.
４．Ikarashi T., Hasegawa H. Detection of monoclonality
in B-cell lymphoma by polymerase chain reaction（PCR）with the use of DNA extraction kit（TakaraDEXPAT）for formalin-fices paraffin-embedded tissues.
Niigata-Ken Koseiren Med J ２０００;１０:１
６-２０. Avail-か らしとしひこ

五十嵐俊彦
目的：病理解剖診断における原因不明の突然死に関して、遺伝子検査による心臓性突然死を確定する。
方法：当施設で１５年間に解剖された４
２５症例中、病理診断が確定できなかった原因不明の突然死７症
例に関して、ナトリウムポンプ α サブユニット遺伝子 SCN５A 変異を検討する。ホルマリン
固定パラフィン切片より回収した DNA 材料を使って、PCR-SSCP 検査を実施した。
成績：４症例に遺伝子変異が示唆され、心臓性突然死と訂正診断できた。
結論：突然死の原因究明において、遺伝子検査は有効である。
キーワード：突然死、原因不明、解剖、心臓性突然死、遺伝子検査、心筋ナトリウムポンプ α サブユ
ニット SCN５A 遺伝子異常、polymerase chain reaction-single strand conformational polymorphism（PCR-SSCP）法―2―

／【Ｋ：】Ｓｅｒｖｅｒ／１５０１０４７９ 新潟県厚生農業協同組合／本文／０４

五十嵐００１‐００４

2015.02.18 19.03.3

Genetic analysis of the myocardial Na+ pump α subunit gene SCN５A mutation in ７ cases of sudden death among our ４２５ autopsy cases - genemutation was suggested in４cases out of７ones by the polymerase chain reaction-single strand conformational polymorphism（PCR-SSCP）method.

Table１. Summary of７cases in this study

Table２. Operational procedure of PCR-SSCP―3―

／【Ｋ：】Ｓｅｒｖｅｒ／１５０１０４７９ 新潟県厚生農業協同組合／本文／０４

厚生連医誌

第２４巻

１号

五十嵐００１‐００４

2015.02.18 19.03.3

１〜４ ２０１５Figure１. A : exon１２-１ PCR-SSCP, B : enlarged view of A, the third band from the top is duplicated in ４th and５th cases,（arrow）, extra band（arrow）, C : exon １８ PCR-SSCP, dislocated bands are shown in ６thcase（arrow）, extra bands are found below the ordinary band cluster in７th case（arrow）. Left side lane
is marker and the first lane（０）was control.
（２０１
４／０５／１
８受付）―4―