Legal Medicine 12 (2010) 305–307

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Legal Medicine

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Case Report

Postmortem genetic analysis for a sudden death case complicatedwith Marfan syndrome

Motonori Takahashi a, Takako Sato b, Minori Nishiguchi a, Koichi Suzuki b, Hajime Nishio a,*

a Department of Legal Medicine, Hyogo College of Medicine, Hyogo 663-8501, Japanb Department of Legal Medicine, Osaka Medical College, Osaka 569-8686, Japan

a r t i c l e i n f o

Article history:Received 19 February 2010Received in revised form 13 May 2010Accepted 11 June 2010Available online 21 August 2010

Keywords:Marfan syndromeFBN1Sudden deathPostmortem genetic analysis

1344-6223/$ - see front matter � 2010 Elsevier Irelandoi:10.1016/j.legalmed.2010.06.003

* Corresponding author. Address: Department of Leof Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyog798 45 6576; fax: +81 798 49 3279.

E-mail address: hnishio@hyo-med.ac.jp (H. Nishio

a b s t r a c t

We report here a sudden death case of a patient previously diagnosed as Marfan syndrome (MFS). Thevictim was dead on the wheel and the cause of death was diagnosed to be a rupture of the thoracic aortaby autopsy findings. MFS is an autosomal dominant disorder of the connective tissue and can be a causeof sudden death. Postmortem genetic analysis revealed a heterozygous p.C1307Y of the FBN1 gene, whichis responsible for pathogenesis of MFS, was evident. This substitution was not found in 400 alleles fromcontrol individuals. In addition, the position 1307 is highly conserved among species. Because the posi-tion 1307 serves as part of the Cys1307-Cys1320 disulfide bond of the fibrillin-1, the p.C1307Y substitu-tion results in loss of the intramolecular disulfide bond. The p.C1307Y substitution may be associatedwith the pathology of the present case, and show a higher risk for aortic rupture and subsequent suddendeath.

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1. Introduction EGF modules (cbEGF). Missense mutations in cbEGF modules are

Marfan syndrome (MFS) is an autosomal dominant genetic dis-order of the connective tissue involving cardiovascular, skeletaland ocular systems. The prevalence of MFS is estimated 2–3 casesper 10,000 individuals [1]. The patients usually show high statureand long fingers along with high arched palate. Gradual aortic dila-tation leading to aortic dissection is one of the most severe compli-cations and often causes sudden death. Mitral valve prolapse is alsosometimes observed.

FBN1, encoding the extracellular connective protein fibrillin-1,is a large and complex gene containing 65 coding exons withapproximately 240 kb in size, therefore the genetic analysis ofFBN1 is not easily performed. Nevertheless, extensive studies havebeen performed and demonstrated approximately 600 mutationsof FBN1 in MFS patients [2]. However, up to the present, a few pa-pers were reported about postmortem genetic analysis on the sud-den death cases complicated with MFS [3,4] or genetic pathology ofMFS [5].

Fibrillin-1 forms intermolecular disulfide bonds with otherfibrillin-1 molecules or other components of the microfibrils, andalso forms intramolecular disulfide bonds. Fibrillin-1 contains 47epidermal growth factor (EGF)-like modules, 43 of which containconsensus sequences for calcium binding, termed calcium-binding

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gal Medicine, Hyogo Collegeo 663-8501, Japan. Tel.: +81

).

the most common mutations found in MFS, and generally affectone of the six highly conserved cysteines or residues in the cal-cium-binding consensus sequences [5].

Here, we describe an autopsy case of sudden death with a FBN1mutation in a patient previously diagnosed as MFS. We also pro-pose the possible molecular mechanism of the founded mutationin the pathogenesis of this case.

2. Case report

2.1. Case history

A Japanese male in his forties diagnosed as MFS had undergonereplacement of the aortic valve, ascending aorta and aortic arch.However, his antemortem clinical data and family histories werenot fully available. He was found dead on the driving seat of hismotor vehicle which had a traffic accident. Despite resuscitationattempts, his death was confirmed at an emergency hospital. Anautopsy was performed approximately 14 h postmortem.

2.2. Autopsy findings

He was 184 cm in height and 70 kg in weight with long fingers.A subcutaneous hemorrhage was found at the left forehead and asmall linear exfoliation was observed on his left arm. No othersubcutaneous hemorrhage, exfoliation and bone fracture wereobserved on his chest, abdomen and back. The ascending aorta

Fig. 2. Identification of the p.C1307Y substitution in FBN1. The DNA sequences ofthe 32nd exons were amplified from the present case (A) and control subjects (B). Aheterozygous transition, c.3920G>A, was observed at the second base in codon 1307of the FBN1 gene. Sense-strand sequences are shown.

306 M. Takahashi et al. / Legal Medicine 12 (2010) 305–307

and aortic arch were replaced by artificial grafts and the left lungwas highly adhered to the thoracic wall. Approximately 3000 mLof blood with a clot was found in the right thoracic cavity. Thedescending thoracic aorta was dilated and had a dissection witha rupture near the diaphragm (Fig. 1). All his organs were anemic.No significant disorders were found in the cardiac valves. No skullfractures, intracranial hemorrhages and macroscopic brain injurieswere found.

No ethanol was detected in both blood and urine using head-space gas chromatography. Toxicological screening tests using aTriage Drugs of Abuse Panels (Biosite Inc., San Diego, CA) producednegative results.

From the autopsy findings, the cause of his death was diagnosedas hypovolemic shock caused by aortic rupture before the trafficaccident.

3. Mutation analysis

Genomic DNA was prepared from blood lymphocytes using aDNA extractor WB kit (Wako Pure Chemical Industries, Kyoto, Ja-pan). All the coding regions of the FBN1 gene (Gene ID; 2200) wereamplified by polymerase chain reaction (PCR) using previously de-scribed primers [6,7]. The amplicons were directly sequencedusing a genetic analyzer (Applied Biosystems, Foster City, CA).

The 32nd exons of FBN1 in the present case and 200 control Jap-anese individuals were amplified by PCR, followed by meltingcurve analysis with a high resolution melting instrument (HR-1;Idaho Technology, Salt Lake City, UT).

This study was approved by the Ethical Committee for Researchof the Human Genome of Hyogo College of Medicine.

4. Results

The DNA sequencing revealed a heterozygous missense transi-tion, p.C1307Y (Fig. 2), and other non-synonymous and synony-mous polymorphic substitutions, p.C472Y (rs4775765), p.N625N(rs25458) and p.S1147S (rs140598). Mutation testing of his familymembers was not available. The p.C1307Y substitution was notfound at least in 400 alleles from control individuals.

Fig. 1. Autopsy findings of the sudden death case complicated with Marfan syndrome. (Acavity held approximately 3000 mL of blood with a clot. (B) The heart and total aorta. T

5. Discussion

The found substitution is already reported in Universal Muta-tion Database without detail information on the case [2]. Althoughmany mutations have been identified in MFS patients by clinically-orientated studies, there are only a few reports of postmortem ge-netic analysis in the sudden death cases of the MFS patients [3,4].Postmortem genetic analysis may give us information about themutations, which possess high risk for sudden death in MFS.

The cysteine at position 1307 in cbEGF module number 17 ishighly conserved among orthologs and serves as part of theCys1307-Cys1320 disulfide bond. This cbEGF module contains acryptic cleavage site for trypsin at Lys1317 at its C-terminal [5].In spite of the diagnosis and following operations, the victim wasdead on the wheel. This case suggests that the Cys1307-Cys1320disulfide bond may be essential for functional importance of thismolecule. Of interest, the mutations of Cys1320 in FBN1 have beenpreviously reported in MFS patients [8,9] and one of these muta-tions caused a dramatic increase in susceptibility to in vitro prote-olysis by trypsin [5]. The amino acid substitution of cysteine to

) Front view of the thoracic cavities (after removal of the sternum). The right thoraciche descending thoracic aorta was dilated with a rupture (white arrows).

M. Takahashi et al. / Legal Medicine 12 (2010) 305–307 307

tyrosine at position 1307 leads to loss of the Cys1307-Cys1320disulfide bond, which may have caused the domain misfoldingand an insufficient structure for maintaining extracellular stability,leading to aortic rupture and sudden death in this case.

Unfortunately, information of relatives of the deceased was notavailable in this case, and further genetic examination and coun-seling of the relatives could not be performed. Taking the possiblepathogenesis of the p.C1307Y substitution into consideration, itscarriers should be extensively cared for possible onset of aortic dis-section and sudden death. The postmortem genetic analyses forheritable disorders should be performed not only to examine thecause of death for the deceased but also to give us an opportunityfor improved prognoses of the mutation carriers [10–13].

Acknowledgment

This study was supported by a Grant-in-Aid for Researchersfrom Hyogo College of Medicine.

References

[1] Judge DP, Dietz HC. Marfan’s syndrome. Lancet 2005;366:1965–76.[2] Béroud C, Collod-Béroud G, Boileau C, Soussi T, Junien C. UMD (Universal

mutation database): a generic software to build and analyze locus-specificdatabases. Hum Mutat 2000;15:86–94.

[3] Klintschar M, Bilkenroth U, Arslan-Kirchner M, Schmidtke J, Stiller D. Marfansyndrome: clinical consequences resulting from a medicolegal autopsy of acase of sudden death due to aortic rupture. Int J Legal Med 2009;123:55–8.

[4] Ripperger T, Tröger HD, Schmidtke J. The genetic message of a sudden,unexpected death due to thoracic aortic dissection. Forensic Sci Int2009;187:1–5.

[5] Booms P, Tiecke F, Rosenberg T, Hagemeier C, Robinson PN. Differential effectof FBN1 mutations on in vitro proteolysis of recombinant fibrillin-1 fragments.Hum Genet 2000;107:216–24.

[6] Körkkö J, Kaitila I, Lönnqvist L, Peltonen L, Ala-Kokko L. Sensitivity ofconformation sensitive gel electrophoresis in detecting mutations in Marfansyndrome and related conditions. J Med Genet 2002;39:34–41.

[7] Nijbroek G, Sood S, McIntosh I, Francomano CA, Bull E, Pereira L, et al. Fifteennovel FBN1 mutations causing Marfan syndrome detected by heteroduplexanalysis of genomic amplicons. Am J Hum Genet 1995;57:8–21.

[8] Liu WO, Oefner PJ, Qian C, Odom RS, Francke U. Denaturing HPLC-identifiednovel FBN1 mutations, polymorphisms, and sequence variants in Marfansyndrome and related connective tissue disorders. Genet Test 1997–1998;1:237–42.

[9] Arbustini E, Grasso M, Ansaldi S, Malattia C, Pilotto A, Porcu E, et al.Identification of sixty-two novel and twelve known FBN1 mutations ineighty-one unrelated probands with Marfan syndrome and otherfibrillinopathies. Hum Mutat 2005;26:494.

[10] Nishio H, Iwata M, Suzuki K. Postmortem molecular screening for cardiacryanodine receptor type 2 mutations in sudden unexplained death: R420Wmutated case with characteristics of status thymico-lymphaticus. Circ J2006;70:1402–6.

[11] Nishio H, Iwata M, Tamura A, Miyazaki T, Tsuboi K, Suzuki K. Identification of anovel mutation V2321M of the cardiac ryanodine receptor gene of suddenunexplained death and a phenotypic study of the gene mutations. Leg Med(Tokyo) 2008;10:196–200.

[12] Nishio H, Kuwahara M, Tsubone H, Koda Y, Sato T, Fukunishi S, et al.Identification of an ethnic-specific variant (V207M) of the KCNQ1 cardiacpotassium channel gene in sudden unexplained death and implications from aknock-in mouse model. Int J Legal Med 2009;123:253–7.

[13] Nishio H, Sato T, Fukunishi S, Tamura A, Iwata M, Tsuboi K, et al. Identificationof malignant hyperthermia-susceptible ryanodine receptor type1 gene (RYR1)mutations in a child who died in a car after exposure to a high environmentaltemperature. Leg Med (Tokyo) 2009;11:142–3.