博 士 （ 医 学） 堀 江 幸 弘
学 位論 文題 名●Seekingfor new diseasesusc
eptibility genes inVogt― Koyanagi− Harada disease（原田病における新しい疾患感受性遺伝子の探求）

学 位論文 内容の要旨

OBJECTIVES: Vogt‑Koyanagi‑Harada (VKH) disease is one of the most frequent formsof uveitis in Japan, and is characterized as a panuveitis accompanied by neurologicallesions such as headache andpleo
cytos
is ofthecereb
rospi
nal fluid,skinlesi
ons such as
vitiligo, alopecia, and inner ear disturbances. This disease is considered to be anautoimmune disease against melanocytes. In earlier studies, melanocyte contains
tyrosinase (TYR), tyrosmase ‑ related protein (TRP) 1 and TRP2, and lymphocytes
separated from VKH disease patients were reactive to peptide fragments derived fromthese proteins. In experimental animal models, melanocyte ‑ specific proteins, TRP1and TRP2, induced an experimental autoimmune disease in Lewis rats that resembled
human VKH disease. Inflammation induced by TRPl in Akita dogs also resembled
human VKH disease. These studies suggest that TYR, TRPl and TRP2 are responsible
for human VKH disease. Interferon‑'r (IFN‑ T) is significantly elevated inthe aqueoushumor and sera of VKH patients. IFN‑ T is important cytokine for VKH disease and isassociated with the development of Thl‑dominant, cell mediated immune responses
which may thereby enhance the expression of human leukocyte antigen (HLA) class IIantigens. VKH disease is known to be associated with HLA class n antigen,HLA‑DRBl*04 and numerous studies have shown that about 80% of VKH patients have
HLA ‑ DRBl*0405. However, little is known about the true pathogenic gene related toVKHdisea
se. As fortheinves
tigat
ion of diseasesusce
ptibi
litygenes, ass
ociat
ion s
tudiesare now primarily conducted with single nucleotide polymorphisms or microsatellites
because they are ubiquitous in the genome. Microsatellite (MS) polymorphisms show agreater diversity than single nucleotide polymorphisms (SNPs) and have been widelyused in both linkage and association studies of disease. Microsatellite linkage
disequilibrium (LD) length is in the approximately 100 kb range when compared withthe shorter range for SNPs.Therefore,theadvan
tageof microsa
tellite analysis isthata
collection of relatively small numbers of polymorphic markers can make association
analyses an immediate reality. To investigate whether melanocyteconta
iningprot
ein isresponsible for VKH disease or not, we analyzed polymorphisms in MSs among TYR,TYRPl and TYRP210cus. The TYR gene, TYRPl gene and TYRP2 gene are located on
the chromosome llq14‑q21, 9q23 and 13q32, respectively. These genes encode theenzymes involved in melanin formation and are expressed specifically in melanocytes.
We speculated that polymorphisms within the TYR, TYRPl and TYRP2 may be related
to VKH disease. As previously described, these candidate genes encode the enzymes
involved in the melanin formation, and are the class of genes thathavebeenasso
ciatedwith depigmentation and ocular developmental defects. In previous studies, mutationsof TYR and TYRPl caused oculocutaneous albinism (OCA) 1, OCA3, andmicrophthalmia, and TYRP2 gene caused melanoma and vitiligo. These mutations
include missense, nonsense, frameshift, and splice site mutations, and deletion of theentire coding sequence.
− 280ー

The IFN T gene on the chromosome 12q24.1 spans approximately 5.4 kb and contains
four exons.Likeother cytokines, the IFN‑T codingre
gi
onisi
nv
ar
iant,wit
hnore
portedpolymorphisms. Single nucleotide polymorphism (SNP; rs2430561) and microsatellite(rs3138557)within the first intronof the IFN‑7gene correlatew
ithahighamountofinv
itroprodu
ctionof I
FN‑ Tand.
are associatedwithdisea
se severityorresistancetodrugtherapy in various autoimmune diseases. This allele is associated with a hig
her oralower risk of a variety of diseases including autoimmune and chronic inflammatoryconditions. The association between SNP (rs2430561) alleles T to A with a low (AA),medium (AT), and high (TT) production of cytokines has been reported in vitro. We
hypothesized that a common allelic variation in these potential functional
polymorphisms may be involved in Thl‑mediated autoimmune diseases, such as VKHdisease.
In this stu
dy ofJapa
nesesubje
cts,we investi
gatedwhet
her t
he microsa
telli
te orsingle
nucleotide polymorphisms in the TYR, TYRPl, TYRP2, IFN‑ r gene and HLA‑DRB1contri
buteto the developm
ent r
isk of VKHdisease and tosomeo
ft
hecli
nic
alf
eat
uresoft
he disease.
METHODS: The study involved totally 136 VKH patients and 176 healthy controls whowere genotyped for HLA‑DRBl, TYR, TYRPl, TYRP2 and IFN.;r. HLA‑DRB1
genotyping was performed by the PCR‑restriction fragment length polymorphism(RFLP) method. Microsatellite (MS) polymorphisms within TYR, TRPl, TRP2 andIFN‑ r and functional single nucleotide polymorphism (SNP) of IFN‑ r were analyzed.
In addition, the haplotype frequencies of the microsatellite in TYR, TRPl and TRP2
were also estimated and statistical analysis was performed. Moreover, clinicalmanifestati
ons of thepati
entswerealsoanalyzed.
RESULTS: The age of the patients ranged from 15 t0 80 years (51.9+13.7, mean+SD).
The study group included 65 men (47.8%) and 71 women (52.2%). Based on thediagnostic criteria for VKH disease, 30 cases (22.1%) were cla
ssifi
ed ascomp
leteVKHdisease, 89 cases (65.4%) as incomplete VKH disease, and17 cases (12.5%) asprobable
VKH disease. Both eyes were affected in all patients. Diffusechoro
iditi
s/sta
iningoffluorescein on angiography was observed in all patients. Sunset glow fundus andnummular chorioretinal depigmented scars were observed in 83.9% and 36.1% of thepatients, respectively. Neurological and auditory disorde
rs were observed in90.1%of
the patients: meningitis (e.g., headache and fever) in 79.8%, tinnitus in 53.0%,and
cerebrospinal fluid pleocytosis in 70.0%.Derm
atolo
gic m
anife
stati
ons w
ere observ
ed in2
2.9%of the patients: alo
pecia(6.9%), polios
is (17.6%), andvitiligo(13.
0%).Therewas
no significant difference between 87 VKH patients and 122 healthy controls at sevenmicrosatellites within the region of TYR, TYRPl and TYRP2, nor in haplotype
frequency. There was no significant difference in the allele and ge
notype fre
quenc
ies
between 136 VKH patients and 176 healthy controls in the SNP (rs2430561) andm
icrosatell
ites (rs3138557) of IF
N‑ r. In our clinicall
y stratifi
ed analysis, weinvestigated the presence of some of the clinical features, such as disease onset,presence of diffuse choroiditis, sunset glow fundus, depigmented scars, meningitis,tinnitus, cerebrospinal fluid pleocytosis, and integumentary finding. None of theseclinical findings were significantlyasso
ciated wit
h themicr
osate
lliteandSNPs.Then
we classified VKHpati
entsaccor
dingto the dia
gnost
ic cr
itenaas comple
te, incomp
lete,and probable VKH disease. However, significant association was not detected between
any type of VKH disease and healthy controls, neither. The magnitude of LD between
SNP (rs2430561) and microsatellite (rs3138557) showed an extremely high value, withpair‑wise LD valued at D' >0.96 in controls and >0.99 in the patients. As for theHLA‑DRBl genotyping of the 87 VKH patients and 122 healthy controls, the phenotype
frequency of HLA‑DRBl*04 was significantly increased in VKH patients (n=71, 81.6%)
compared to healthy controls (n=53, 43.4%) [Odds ratio (OR) = 5.78, P= 3.08E‑8, P
c=7.70E‑8]. Within the HLA‑DRBl*04, the phenotype frequency of HLA‑DRBl*0405was
remarkably higher in VKH patients (n=61, 70.1%) compared to healthy controls (n=35,28.7%)(OR= 5.8
3, P= 3.1
0E‑9,Pc =7.90
E‑8).

― 281一CONCLUSIONS: We concludedthatthereisnogeneticsusceptibilityorincreasedrisk
attributed to the TI72, TYRPl, TYRP2 and IFN‑ r. HLA‑DRBl*0405 showed ahighly
significantassociationasexpected.Themutat
ionalchar
acter
izati
onofgenesinv
olved
in VKH disease will provide additional insight into the molecular mechamsms
underlyingthiscommonuveitisintheJapanesepopulation.

― 282 ‑

学 位論文審査の要旨
主 査 教 授 本 間 研 一
副 査 教 授 笠 原 正 典
副 査 教 授 守 内 哲 也

学位論文題名●Seeking for new disea
se susceptibi
lity genesln
Vogt−Koyanagi−Harada disease（原田病における新しい疾患感受性遺伝子の探求）

Vogt‑Koyanagi‑Harada病 （原 田病 ）は 髄膜刺 激症 状、 髄液 細胞 増多 、感 音性 難聴 など の神 経障 害、白 斑、脱毛、白髪などの皮膚障害を伴う而眼性の汎ぶ
ど う膜 炎で ある 。こ の疾患 はメラノサイトに対する自己免疫疾患であると考え
ら れ て い る 。 本 研 究 は 、 HLA‑DR、 TYR、 TRP1、 TRP2そ し て IFN:ッ 遺 伝 子の 多 型 を 原田 病患 者136例と 健常 人174例 で比較 した もの であ る。 北海 道大 学
眼 科あ るい は横 浜市 立大学 眼科 で、 原田 病と 診断 され た患者の末梢血からDNAを 採 取 し 、 Primary chain reaction (PCR)、 ダ レ ク ト シ ー ク エ ン ス 法、HLAーDRB1タイピングにより比較検討した。
その結果、′I'YR、TYRP
1，2、IFNーッ遺伝子では、疾患群と健常群の間で有意差 は み ら れ な か っ た 。 原 田 病 の 抗 原 と考 えら れる TYR、 TYRP1， 2にお ける 遺伝 子レ ベル での 違い はない ことがわかった。IFN‑ッ遺伝子にっいても有意差が見 られ なか った こと から、 IFN‑ッ遺伝子により前房や血中の産生量が増加してい る わ け では ない こと がわ かっ た。 HI」 AーDRタイ ピン グの 結果 、DR4が患 者群 で81.6％ 、健 常対 照群で 43.4％と有意に患者群で多くみられた。そのうち特に DRBl*0405が 患 者 群 で 70.1％ 、 健 常 対 照群で 28.7％、 相対 危険 度5.83、 補正 P値 が 7.9X10の マ イ ナ ス 8乗 と 強 い 相 関 が み ら れ た 。 結 論 と し て 、HLA‑DR4と く に HLA‑DRB1六 0405が 原 田 病 患 者 で 有 意 に 多 く み ら れ た が 、病態 と 関 係 す る と 考 え ら れ て い る TYR、 TRP1、 TRP2、 IFN‑v遺 伝 子 に は 、原田病患者と健常人とに違いはみられなかった。
学 位 論 文の 審査 は、 平成 21年 2月3日、 主査の 本間 教授 、副 査の 笠原 教授 、守 内教 授の 3名に より 、公 開審 査と して 医学 研究 科臨床 大講堂で行われた。なお 、 出 席 者は 約50名で あっ た。 主査 から 紹介が あっ た後 、申 請者 はス ライ ドを 用 い て 約10分間 にわ たり 、学 位論 文の 内容を 発表 した 。そ の後 、主 査、 副

査と申請者による質疑応答が約20分問にわたって行われた。笠原教授からは、今回対象とした遺伝子では有意差が認められなかったが、今後どの遺伝子に着目すべきか、原田病で自己免疫が起こる機序、チロシナーゼ、TRP1，2、IFNのコード領域に韜ける多型にっいて、守内教授からは、研究試料収集法、人種による臨床症状の相違、病態解明の観点から網羅的燈RNA解析の妥当性について、本間教授からは、原田病におけるメラノサイトの抗原部位、本研究以外の病態解明戦略、臨床症状発現に韜ける環境因子について、それぞれ質問があった。申請者は、いずれの質問に対しても、学位論文のデーターや過去の文献を引用し、概ね適切に回答した。
本研究は、本邦に多発する原田病の病態を、分子生物学的および遺伝学的手法 を用 いて解 明す るこ とを 試みた もの で、 HLA‑DR4とく にHLA‑DRBl'0405が原田病患者で多くみられることを明らかにした。審査員一同は、これらの成果を高く評価し、大学院課程における研鑽や取得単位なども併せ申請者が博士（ 医学） の学 位を受 ける のに充 分な 資格を 有するものと判定した。